Plasmid-derived llad II restriction-modification system from lactococcus lactis

ABSTRACT

A number of type II restriction-modification (R-M) systems have been derived from plasmids found in  Lactococcus lactis  strains from the Danish starter culture TK5. The R-M systems LlaAI, LlaBI and LlaDII are claimed with their nucleotide sequences containing open reading frames (ORFs) coding for restriction endonucleases and corresponding methylases. Also a DNA cassette comprising one or more of the R-M systems and fragments thereof in combination with DNA encoding other phage resistance mechanisms is claimed as are cloning and expression vectors including DNA selected from the group consisting of the R-M systems, fragments thereof and DNA cassette, cells transformed with the expression vectors, a method of conferring increased virus resistance on a cell, and the individual restriction endonucleases and methylases of the R-M systems.

This invention relates to plasmid-derived type IIrestriction-modification (R-M) systems from Lactococcus lactis, DNAfragments coding for individual methylases and restriction endonucleasesthereof, DNA cassettes for increasing phage resistance in lactic acidbacteria, cloning and expression vectors including the R-M systems, amethod of conferring increased phage resistance on a lactic acidbacterium, lactic acid bacteria and Lactococcus lactis strains carryingthe expression vectors, as well as methylases and restrictionendonucleases encoded by the R-M systems.

BACKGROUND OF THE INVENTION

Lactococcus strains are used as starter cultures for the production ofcheeses and fermented milks. Bacteriophage infection of the starterculture remains a serious problem for the cheese industry and can resultin a slow or dead cheese vat. Several mechanisms of phage defense havebeen identified in lactococci. These include adsorption blocking,abortive infection, and R-M systems (26). A report has shown thebeneficial effect of using different phage resistance mechanisms inrotation (42). By cloning phage resistance mechanisms from lactococci itwould be possible to construct a “cassette” like system consisting ofdifferent phage resistance mechanisms.

Restriction-modification (R-M) systems have been found in a wide rangeof bacteria. At least three different types of R-M systems, type I, IIand III, have been found and characterized with respect to theirrequirement of Mg²⁺, ATP and S-adenosyl-methionine (2). The type II R-Msystem is by far the most simple and best understood of the R-M systems,containing a separate methylase (MTase) which uses S-adenosyl-methionineas the methyl donor and an endonuclease (ENase), both recognizing thesame sequence. Today more than 200 different type II R-M systems havebeen identified and more than 100 of them have been cloned, mainlybecause of their importance as tools in molecular biology and theimportant knowledge which is achieved of protein-DNA interactions. Thegenetic characterization of type II R-M systems shows that the genes forthe ENase and the MTase are closely located, although not always in thesame orientation. The MTase and the ENase from the same type II R-Msystem normally do not show any homology to each other at the amino acidlevel despite the fact that they recognize the same DNA-sequence.Comparisons between type II MTases have shown that strong similaritiesexist within the group consisting of 5-methylcytosine MTases (m⁵CMTases) and within the group consisting of N4-methylcytosine (m⁴C) andN6-methyladenosine (m⁶A) MTases (29). The m⁵C MTases have about tencommon amino acid sequence motifs, whereas the m⁴C and m⁶A MTases sharetwo major common amino acid sequence motifs. In contrast, the ENaseshave generally very little homology in common, and no strong sequencemotifs have been found.

Several plasmids encoding R-M systems have been found in lactococci.However they have not been characterized at the molecular level, butonly in vivo by their efficiency in restricting phages (38). Fitzgeraldet al. (11) examined eight different strains and found only one type IIendonuclease activity, R, ScrFI, in one strain, L. lactis UC503(originally designated Streptococcus cremoris F). This first type II R-Msystem, ScrFI, isolated and characterized from a L. lactis strain, hasbeen found to be chromosomally encoded (11, 8). Later Daly andFitzgerald (6) examined seven strains more from different startercultures and found that six of the strains had ENase activity similar toR, ScrFI. No other type II ENase activities were found. The ENase fromScrFI recognized 5′-CC↓NGG-3′. Two ScrFI MTase-encoding genes have beencloned and characterized, but neither of the two isolatedM,ScrFI-carrying clones exhibited any ScrFI ENase activity (8). Thenucleotide sequences of the two MTase-encoding genes have beendetermined (8, 45). Both contained all ten of the predictive motifsnormally found in m⁵C MTases. Mayo et al. (32) have reported type IIactivity, LlaI recognizing the sequence CC↓WGG from L. lactis NCDO497,but they did not determine whether it was chromosomally or plasmidencoded.

A type IIS system, LlaI, has been identified on the lactococcal plasmidpTR2030, which also codes for an arbortive infection mechanism (17). Thenucleotide sequence of a type IIS MTase, M,LlaI, from the plasmidpTR2030 has been identified and determined (18).

TK5 is a Danish starter culture, which has been used for the productionof Cheddar cheese since 1982 (33). This starter culture has a markedresistance to phages as the dairy during 11 years of continuousproduction did not observe any delay in acidification due to a phageinfection, even though phages were isolated from the whey. The starteroriginates from an old traditional dairy starter culture, which consistsof an unknown number of L. lactis strains. Sixty-two bacterial isolateswere purified from the TK5 starter. 33 of the isolates were arrangedaccording to their plasmid profiles into six groups of identical ornearly identical profiles; 27 of the isolates showed unique plasmidprofiles (22). All isolates have between 5 and 10 plasmids.

In order to identify plasmid-encoded phage resistance in Lactococcus acotransformation procedure was used. Total plasmid DNA from L. lactisstrain W56 isolated from the TK5 starter culture (33) was transformedtogether with the vector pVS2 (53) into Lactococcus lactis subsp.cremoris MG1614. We selected for the Cm^(R) marker on pVS2.Transformants were tested for increased phage resistance. In this way weidentified three plasmids (pJW563, pJW565, and pJW566) which coded forR-M systems in W56 (23). These plasmids ranged in size from 11 to 25 kb.The efficiency of plating (EOP) for the isometric-headed phage p2 (16)varied from 10⁻² to 10⁻³ for different plasmids. The existence ofmultiple R-M encoding plasmids in Lactococci's strains has previouslybeen reported by Chopin's group in France (5, 13).

That multiple R-M-encoding plasmids can increase phage resistance wasconfirmed by stacking two-three plasmids (23, 24). The data in thefollowing Table A show the efficiency of plating (EOP) of phage p2 onthe various transformants, the numbers in parentheses in column 2 showthe EOP of phage p2 with the R-M plasmid alone in L. lactis MG1614.

TABLE A Plasmid encoding R-M systems assembled in L. lactis and theireffects on the EOP of phage p2 or jj50^(a). Transformants^(b) Plasmidencoding R-M EOP MG1614 none 1  T128 pJW563 (10⁻³) + pJW565 (10⁻²) 10⁻⁵T46 pJW563 (10⁻³) + pJW566 (2 × 10⁻²) 4 × 10⁻⁶ T45 pJW565 (10⁻²) +pJW566 (2 × 10⁻²) 10⁻³ T8  pJW563 (10⁻³) + pJW565 (10⁻²) + 3 × 10⁻⁶pJW566 (2 × 10⁻²) J96 pJW563 (10⁻³) + pFV1001 (10⁻¹) 10⁻⁵ J92 pJW563(10⁻³) + pFV1201 (10⁻¹) 10⁻⁵ J75 pJW563 (10⁻³) + pFV1001 (10⁻¹) + 4 ×10⁻⁷ pTRK12 (10⁻¹) ^(a)Phages p2 and jj50 were propagated on L. lactisMG1614[pVS2]. L. lactis MG1614 is a sm^(R), Opp^(d) derivative of L.lactis MG1363 (46). ^(b)All transformants also harbored pVS2.

As shown in Table A, the effect of assembling R-M plasmids were additivein most cases. This supports the importance of R-M systems in the phageresistance of the TK5 starter. We did not obtain completely phageresistant strains, however, Sing and Klaenhammer (41) showed that incombination with other phage resistance mechanisms, e.g., abortiveinfection, R-M systems are powerful tools.

Transformant T1.1 (L. lacits MG1614+pJW563) and a transformantharbouring a plasmid pFW094 from L. lactis W9 (34) exhibited type IIendonuclease activity showing that type II R-M systems can be plasmidencoded in Lactococcus lactis.

The ENases R.LlaAI and R.LlaBI from W9 and W56, respectively, werepartially purified; and the recognition sequences for both ENases wereidentified by digesting well known DNA (pBR322/328, λ DNA) with therespective ENases, treating the fragments with either the Klenowfragment of DNA polymerase or mung bean nuclease and ligating theresulting fragments into pBluescriptIISK+(Stratagene, La Jolla, Calif.,USA) digested with EcoRV. By sequencing the junction fragments of theobtained clones, the recognition sequence of the respective ENases couldbe determined (34).

We found that R.LlaAI and R.LlaBI recognized 5′-↓GATC-3′ and5′-C↓TRYAG-3′, respectively, digesting as indicated by the arrows. ENaseR.LlaAI is therefore an isoschizomer of MboI from Moraxella bovis andDpnII from Streptococcus pneumoniae. R.LlaBI is an isoschizomer of SfcIfrom Enterococcus faecium. Identical ENase cleavage patterns wereobtained in digests of pBluescriptIISK+, pBR322 and M13mp20 with R.LlaAIand MboI, and with R.LlaBI and SfcI, respectively.

SUMMARY OF THE INVENTION

Attempts to clone LlaAI or LlaBI in Escherichia coli by screening thetransformants for increased phage resistance to λvir were unsuccessful.However, it was possible to clone and subclone the LlaAI, LlaBI andLlaDII R-M systems directly in Lactococcus (see the following examples).When fragments from LlaAI and LlaDII were later transferred to E. coli,only the MTase activity was expressed, while ENase activity could not bedetected. It was not possible to clone the entire LlaBI system or thegene encoding the methylase in E. coli.

From the nucleotide sequence of LlaAI we could identify three ORFs,transcribed in the same direction and coding for putative proteins of284, 269 (or 257) and 304 amino acids. By comparison of the deducedamino acid sequences with data in EMBL and GenBank we found that two ofthe proteins had about 80% homology to the two MTases from the DpnII R-Msystem, while the third had about 30% homology to the correspondingENase. This indicates that the LlaAI R-M system consisted of twoputative MTases and one putative ENase. Based on the observation thatthe two ENases, R.LlaAI and DpnII, recognize the same nucleotidesequence, 5′-GATC-3′, that both have two MTases, that the two MTases ofDpnII methylate adenine (9), and that the LlaAI ENase is sensitive tomethylated DNA from dam⁺ E. coli strain, we conclude that the LlaAIMTases most probably methylate adenine. It has been suggested that thepreviously sequenced lactococcal MTases, M,LlaI and M,ScrFI, methylateadenine and cytosine, respectively (18, 8).

From the nucleotide sequence of LlaBI we could identify two ORFs withpredicted proteins of 580 and 299 amino acids. They are transcribeddivergently. We did not find any very strong homology to other ENases orMTases in the EMBL and GenBank.

Accordingly, in a first aspect the present invention provides aplasmid-derived type II restriction-modification (R-M) system, termedLlaAI, from Lactococcus lactis subsp. cremoris W9, said system encodingat least one methylase and a restriction endonuclease with therecognition sequence 5′-↓GATC-3′, characterized in that the systemcomprises

i) an open reading frame, termed ORF1, from nucleotide 769 to nucleotide1620 in the enclosed SEQ ID No. 1, coding for a methylase, termedM.LlaAIA, having the amino acid sequence shown in the enclosed SEQ IDNo. 2;

ii) an open reading frame, termed ORF2, from nucleotide 1613 tonucleotide 2419 in the enclosed SEQ ID No. 3 (same as SEQ ID No. 1) orfrom nucleotide 1649 to nucleotide 2419 in the enclosed SEQ ID No. 5(same as SEQ ID No. 1), coding for a methylase, termed M.LlaAIB, havingthe amino acid sequence shown in the enclosed SEQ ID No. 4 or SEQ ID No.6, respectively; and

iii) an open reading frame, termed ORF3, from nucleotide 2412 tonucleotide 3323 in the enclosed SEQ ID No.7 (same as SEQ ID No. 1),coding for a restriction endonuclease, termed R.LlaAI, having the aminoacid sequence shown in the enclosed SEQ ID No. 8.

This aspect of the invention also includes DNA fragments comprising eachof the ORFs in the R-M system LlaAI, i.e.

a) A DNA fragment coding for a methylase, termed M.LlaAIA, said fragmentcomprising the DNA sequence from nucleotide 769 to nucleotide 1620 inthe enclosed SEQ ID No. 1.

b) A DNA fragment coding for a methylase, termed M.LlaAIB, said fragmentcomprising the DNA sequence from nucleotide 1613 to nucleotide 2419 orfrom nucleotide 1649 to nucleotide 2419 in the enclosed SEQ ID No. 1.

c) A DNA fragment coding for a restriction endonuclease, termed R.LlaAI,said fragment comprising the DNA sequence from nucleotide 2412 tonucleotide 3323 in the enclosed SEQ ID No. 1.

In a second aspect the present invention provides a plasmid-derived typeII restriction-modification (R-M) system, termed LlaBI, from Lactococcuslactis subsp. cremoris W56, said system encoding at least one methylaseand a restriction endonuclease with the recognition sequence5′-C↓TRYAG-3′, characterized in that the system comprises

i) an open reading frame, termed ORF1, from nucleotide 422 to nucleotide2161 in the enclosed SEQ ID No. 9, coding in the complementary strandfor a methylase, termed M.LlaBI, having the amino acid sequence shown inthe enclosed SEQ ID No. 10; and

ii) an open reading frame, termed ORF2, from nucleotide 2464 tonucleotide 3360 in the enclosed SEQ ID No. 9, coding for a restrictionendonuclease, termed R.LlaBI, having the amino acid sequence shown inthe enclosed SEQ ID No. 11.

This aspect of the invention also includes DNA fragments comprising eachof the ORFs in the R-M system LlaBI, i.e.

d) A DNA fragment coding in the complementary strand for a methylase,termed M.LlaBI, said fragment comprising the DNA sequence fromnucleotide 422 to nucleotide 2161 in the enclosed SEQ ID No. 9.

e) A DNA fragment coding for a restriction endonuclease, termed R.LlaBI,said fragment comprising the DNA sequence from nucleotide 2464 tonucleotide 3360 in the enclosed SEQ ID No. 9.

In a third aspect the present invention provides a plasmid-derived typeII restriction-modification (R-M) system, termed LlaDII, fromLactococcus lactis subsp. cremoris W39, said system encoding at leastone methylase and a restriction endonuclease, characterized in that thesystem comprises

i) an open reading frame, termed ORF1, from about nucleotide 743 tonucleotide 1282 in the enclosed SEQ ID No. 12, coding for a restrictionendonuclease, termed R.LlaDII, having the amino acid sequenceessentially as shown in the enclosed SEQ ID No.13 and with therecognition sequence 5′-GC↓NGC-3′, and

ii) an open reading frame, termed ORF2, from nucleotide 1391 tonucleotide 2341 in the enclosed SEQ ID No. 12, coding for a methylase,termed M.LlaDII, having the amino acid sequence shown in the enclosedSEQ ID No. 14.

This aspect of the invention also includes DNA fragments comprising eachof the ORFs in the R-M system LlaDII, i.e.

f) A DNA fragment coding for a restriction endonuclease, termedR.LlaDII, said fragment comprising the DNA sequence from aboutnucleotide 743 to nucleotide 1282 in the enclosed SEQ ID No. 12.

g) A DNA fragment coding for a methylase, termed M.LlaDII, said fragmentcomprising the DNA sequence from nucleotide 1391 to nucleotide 2341 inthe enclosed SEQ ID No. 12.

Further, the invention includes a DNA cassette comprising one or more ofthe R-M systems and DNA fragments according to the invention incombination with DNA encoding other phage resistance mechanisms selectedfrom the group consisting of adsorption blocking, abortive infection andR-M systems.

The invention also provides a cloning vector including DNA selected fromthe group consisting of R-M systems, DNA fragments and DNA cassettesaccording to the invention, and more specifically the plasmid pSNA1introduced in Lactococcus lactis MG1614 and deposited under theaccession number LMG P-15720, the plasmid pAG55 introduced inLactococcus lactis MG 1614 and deposited under the accession number LMGP-15719, and the plasmid pCAD1 introduced in Lactococcus lactis subsp.cremoris LM2301 and deposited under the accession number LMG P-16901.

The invention also provides an expression vector including DNA selectedfrom the group consisting of R-M systems, DNA fragments and DNAcassettes according to the invention under the control of a promotercapable of providing expression thereof in a host cell, particularly aGram-positive bacterium, and more particularly a lactic acid bacterium,especially Lactococcus lactis.

Further, the invention provides a method of conferring increased virusresistance on a cell wherein said cell is transformed with an expressionvector according to the invention. In particular the invention providesa method of conferring phage resistance on a Gram-positive bacterium,more particularly a lactic acid bacterium, and especially a Lactococcuslactis strain, wherein said bacterium is transformed with an expressionvector according to the invention. The invention also comprises a cell,particularly a Gram-positive bacterium, more particularly a lactic acidbacterium, and especially a Lactococcus lactis strain, which carries anexpression vector according to the invention.

In addition, the invention provides:

a methylase, termed M.LlaAIA, having the amino acid sequence shown inthe enclosed SEQ ID No. 2;

a methylase, termed M.LlaAIB, having the amino acid sequence shown inthe enclosed SEQ ID No. 4 or SEQ ID No. 6;

a restriction endonuclease, termed R.LlaAI, with the recognitionsequence 5′-↓GATC-3′, said endonuclease having the amino acid sequenceshown in the enclosed SEQ ID No. 8;

a methylase, termed M.LlaBI, having the amino acid sequence shown in theenclosed SEQ ID No. 10;

a restriction endonuclease, termed R.LlaBI, with the recognitionsequence 5′-C↓TRYAG-3′, said endonuclease having the amino acid sequenceshown in the enclosed SEQ ID No. 11;

a restriction endonuclease, termed R.LlaDII, with the recognitionsequence 5′-GC↓NGC-3′, said endonuclease having the amino acid sequenceessentially as shown in the enclosed SEQ ID No. 13; and

a methylase, termed M.LlaDII, having the amino acid sequence shown inthe enclosed SEQ ID No. 14.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Maps, restriction and modification activities of pFW094 andsubclone fragments thereof and the products of the LlaAI genes. pFW094is a wild-type plasmid and is shown linearised by cleavage at a XhoIsite. Arrows indicate the position of the putative open reading framesand the direction of transcription. The putative sizes in amino acidsare indicated for the open reading frames.

FIG. 2. Maps, restriction and modification activities of pJW563, itsderivatives and the subclone fragment in pSNB1 as well as the productsof the LlaBI genes. pJW563 is a wild-type plasmid. Plasmids are shownlinearised by cleavage at a XhoI site. Arrows indicate the position ofthe putative open reading frames and the direction of transcription. Theputative sizes in amino acids are indicated for the open reading frames.The figure illustrates the activity measured for the different plasmidsconstructed. A: restriction map of pJW563; B: effect on R-M activity byintroducing a chloramphenicol resistance cassette in the ClaI site atposition 11 kbp, C: effect on R-M activity by deletion of the 1.2 kbpBclI fragment; D effect on R-M activity by introducing an erythromycinresistance cassette in the BglII site at position 9.2 kbp; and E:cloning of the 4.0 kbp HindIII fragment. Abbreviations: Bc, BclI; Bg,BglII, E, EcoRI; H, HindIII X, XhoI.

FIG. 3. Restriction map of plasmid pHW393.

FIG. 4. Agarose gel (0,8%) showing the restriction patterns obtained bycleaving different DNA with: BsoFI (lane 1 and 3) and LlaDII (lane 2 and4). Lanes 1 and 2: pSA3; Lanes 2 and 4: pBluescript IISK+.

DETAILED DESCRIPTION OF THE INVENTION

In order to determine the biological diversity of the R-M systems wecompared six plasmids isolated in our laboratory with three isolated inChopin's laboratory and one from Klaenhammer's laboratory by use of theprolate headed phage c2 (25). Phage c2 was propagated on eachplasmid-containing strain and tested for restriction by all strains. Theresults are assembled in the following Table 1.

TABLE 1 The EOP of phage c2 on L. lactis strains carrying different R-Mencoding plasmids. Phage c2 propagated on strain T1.1^(a) T21.5^(d)T46.22^(a) T3442.7^(a) T912.2^(a) T2235.5^(a) TB123^(b) IL1420^(c)IL1530^(c) IL181 MG1614 pJW563 pJW565 pJW566 pFV0802 pFV1001 pFV1201pTRK12 pIL6 pIL7 pIL10 Plasmid none W56 W56 W56 T29W5 V32.2 KH NCK40IL594 IL594 IL96 from isolate EOP^(d) MG1614 1 1 1 1 1 1 1 1 1 1 1 T1.13 × 10⁻¹ 1 3 × 10⁻¹ 3 × 10⁻¹ 10⁻¹ 10⁻¹ 2 × 10⁻¹ 1 4 × 10⁻¹ 2 × 10⁻¹ 4 ×10⁻¹ T21.5 8 × 10⁻⁴ 4 × 10⁻³ 1 7 × 10⁻³ 10⁻³ 10⁻³ 7 × 10⁻³ 4 × 10⁻² 2 ×10⁻³ 5 × 10⁻⁴ 2 × 10⁻³ T46.22 5 × 10⁻³ 9 × 10⁻³ 5 × 10⁻³ 1 1 3 × 10⁻³ 3× 10⁻³ 7 × 10⁻³ 4 × 10⁻³ 5 × 10⁻³ 10⁻² T3442.7 10⁻² 8 × 10⁻³ 6 × 10⁻³ 11 10⁻² 6 × 10⁻³ 10⁻² 7 × 10⁻³ 6 × 10⁻³ 10⁻² T912.2 9 × 10⁻² 3 × 10⁻¹ 2 ×10⁻¹ 4 × 10⁻¹ 4 × 10⁻¹ 1 2 × 10⁻¹ 5 × 10⁻² 4 × 10⁻² 10⁻¹ 10⁻¹ T2235.5 6× 10⁻² 8 × 10⁻² 2 × 10⁻¹ 9 × 10⁻² 10⁻¹ 8 × 10⁻² 1 3 × 10⁻¹ 8 × 10⁻² 7 ×10⁻² 10⁻¹ TB123 4 × 10⁻² 3 × 10⁻² 10⁻¹ 9 × 10⁻² 10⁻¹ 4 × 10⁻² 10⁻² 110⁻¹ 5 × 10⁻² 10⁻¹ IL1420 7 × 10⁻⁴ 2 × 10⁻⁴ 2 × 10⁻⁵ 5 × 10⁻⁴ 2 × 10⁻⁴10⁻⁴ 7 × 10⁻² 3 × 10⁻⁶ 7 × 10⁻² 2 × 10⁻⁵ 4 × 10⁻⁴ IL1530 6 × 10⁻³ 6 ×10⁻³ 2 × 10⁻³ 6 × 10⁻³ 10⁻³ 9 × 10⁻⁴ 10⁻³ 8 × 10⁻³ 10⁻¹ 10⁻¹ 4 × 10⁻³IL1813 3 × 10⁻⁵ 10⁻⁴ 4 × 10⁻⁵ 4 × 10⁻⁵ 10⁻⁵ 2 × 10⁻⁵ 3 × 10⁻⁵ 7 × 10⁻⁵ 2× 10⁻⁵ 3 × 10⁻⁶ 1 Type-II^(e) — +(LlaBI) — — — — — — — — — ^(a)Theplasmids were cotransformed into L. lactis MG1614 together with pVS2 andafterwards cured for pVS2 by growing the cells in the presence ofnovobiocin. ^(b)A gift from T. Klaenhammer. The plasmid is in L. lactisMG1363. ^(c)A gift from M.-C. Chopin. The plasmids are in L. lactisIL1403. (5). ^(d)EOP was calculated as the phage titer on the teststrain divided by the titer on L. lactis MG1614. The EOP is an averageof at least four experiments. ^(e)Type-II ENase activity was carried outas written in FIG. 1. +. indicate activity and −. no activity.

The results in Table 1 demonstrate that phages propagated on T46.22[pJW566] and T3442.7 [pFV0802] were not restricted by each other. Thisshowed that pJW566 and pFV0802 probably code for identical R-M systems.T1.1 did not restrict phages propagated on TB123, but TB123 restrictedT1.1 propagated phages. This indicated either that two R-M systems couldbe encoded on pTRK 12, one of which is identical to the one encoded bypJW563, or that the MTase encoded by pTRK12 cross-protects against thepJW563-encoded R-M system. We also observed that phage c2 propagated onIL1420 or IL1530 gave a lower titer on the propagating host, than on theplasmid-free strain L. lactis MG1614. This is not due to differences instrain background, since this effect was not found in L. lactis IL1813.These data imply that in addition to a R-M system, pIL6 and pIL7 alsocode for an additional phage resistance mechanism, not previouslyidentified. The remaining MTases were not able to protect the phage fromrestriction by the other R-M systems, indicating that they are differentfrom each other. This shows that L. lactis strains have a largediversity of plasmid-encoded R-M systems.

The diversity of R-M-encoding plasmids identified in Lactococcus made usscreen all isolates from the TK5 starter for the presence of type IIENase activity. Five different type II ENase activities, R.LlaAI,R.LlaBI, R.LlaCI, R.LlaDI and R.LlaEI, were identified. Table 2 depictsthe distribution of ENase activity among the TK5 isolates.

TABLE 2 Type-II ENase activity in L. lactis isolates from the TK5starter culture. Type II ENase activity^(b) R. LlaAI R. LlaBI R. LlaCIR. LlaDI R. LlaEI Group^(a) L. lactis strains 1: (16) W21, W70 2: (4) 3:(5) W9, W25, W69, W71, W72 4: (5) W3, W52, W56, W66, W67 5: (4) W41, W436: (2) W40 outside: W14, W53, W15 W39 W12 (29) W54 ^(a)The isolates weregrouped according to their plasmid profile (22). Numbers in parenthesesshow the number of isolates belonging to the group.

It is seen from Table 2 that none of the screened isolates expressedmore than one type II ENase activity. The most common ENase was theR.LlaAI, which was found in 12 (19 %) of the isolates. R.LlaBI was foundin five (8%), R.LlaCI in two (3%), with R.LlaDI and R.LlaEI in one (2%)of the isolates. Thus, approximately ⅓ of the strains in the TK5 starterculture contained a type II R-M system.

From two of the strains that showed type II ENase activity, W9 (Group 3)and W56 (Group 4), we had previously isolated R-M-encoding plasmids.This enabled us to test if the activity was chromosomally or plasmidencoded. The transformants T1.1[pJW563], T21.5[pJW565], T46.22[pJW566](Table 1) carried plasmids from W56; TW093[pFW093] and TW094[pFW094]carried plasmids from W9. These were examined and we found that onlyT1.1 and TW094 exhibited type II ENase activity. The two systemsidentified in the TK5 isolates W12 and W15 quite likely are plasmidencoded. In order to determine whether the LlaDI endonuclease wasplasmid encoded, the cotransformation procedure into L. lactis MG1614was made with total plasmid DNA from W39.We found that only transformantL. Iactis MG1614[pHW393] expressed LlaDI endonuclease activity whichshows that also the LlaDI R-M system is plasmid encoded. The othertransformants from Table 1 were also examined. None of these strainsexhibited type II ENase activity. This suggests that other ENaseactivity than type II exists in Lactococcus. Additional experiments arerequired to determine the type of R-M systems coded for on theseplasmids.

The recognition sequences for LlaCI, LlaDI and LlaEI have not yet beendetermined. It is interesting that we have found a much wider diversityof type II ENases in Lactococcus, than previously reported (27). Alsothe two ENases characterized by us have recognition sequences with atleast 50% A+T, in contrast to the 5 bp-recognizing ENases (5′-CCNGG-3′or 5′-CCWGG-3′) reported for Latococcus and Streptococcus thermophilus(27). This may have practical implications, as Lactococcus andlactococcal phages have approximately 60% A+T in their genomes (40).

When we tried to clone the LlaDI R-M system we discovered that theplasmid pHW393 also coded for another type II R-M system which wedesignated LlaDII.

The cloning and sequencing of the R-M systems LlaAI, LlaBI and LlaDII isdescribed in the following Examples 1, 2 and 3, respectively.

EXAMPLE 1

In the following we describe the identification, cloning and sequence ofthe plasmid-derived LlaAI R-M system from Lactococcus lactis subsp.cremoris W9. We show that the plasmid-free Lactococcus strain MG1614obtains a higher degree of phage resistance with the plasmid pFW094 orthe plasmid pSNA1 than without it. The cloning and sequence of the LlaAIR-M system is shown, and the putative ORFs and orientation of two MTasesand one ENase are shown. The deduced amino acid sequences were comparedwith known type II R-M systems and, surprisingly, strong homology wasfound to the isoschizomer DpnII R-M system from Diplococcus pneumoniaeand to MboI from Morexella bovis.

Materials and Methods

Strains, phages, plasmids and growth condition. Lactococcus lactissubsp. cremoris (L. lactis) W9 obtained from E. Waagner Nielsen (22),and Lactococcus lactis subsp. cremoris MG1614 (12) (previouslydesignated L. lactis subsp. lactis MG1614 (14)), obtained from Atte vonWright, was grown at 30° C. in M17 medium (44) supplemented with 0,5%glucose (GM17). When required, 10 μg/ml chloramphenicol or 5 μg/mlerythromycin was added. The Escherichia coli strains, XL1-Blue(Stratagene) and TC1685. obtained from Tove Atlung, were grown at 37° C.in LB supplemented with chloramphenicol, erythromycin, tetracycline orampicillin at the concentrations 10, 100, 12,5 and 100 μg/ml,respectively. Plasmid pVS2 was cured with 1 μg/ml novobiocin. Theisometric headed phages jj50 (23) and c2 (25) were propagated and plaqueassayed as described by Terzaghi and Sandine (44). Phage sensitivity wasperformed by plaque assay and cross streaking with phage jj50 asdescribed previously (23). Phage λ b2 was propagated as described bySambrook et al (37). The plasmids used in this study are shown in Table3.

TABLE 3 Plasmids. Source Plasmid Relevant characteristics or referencepFW094 15.5 kbp isolated from L. lactis this work W9 pVS2 5.0 kbp,Cm^(R), Ery^(R) A. von Wright pSA3 shuttle vector, Ery^(R) inLactococcus (7) Cm^(R) in E. coli pBluescriptIISK+ Am^(R) StratageneName Name cloned in cloned in pSA3 pIISK+ pSNA1 pNA1 6.0 kbp EcoRVfragment of this work pFW094 pSNA2 pNA2 5.5 kbp EcoRV-HaeIII fragment ofthis work pSNA1 pSNA3 pNA3 3.1 kbp Bg/II-Sau3 A fragment of this workpSNA1 pSNA4 pNA4 4.6 kbp Bg/II-BstXI fragment of this work pSNA1 pSNA5pNA5 2.1 kbp Sau3A-EcoRV fragment of this work pSNA1 pSNA6 pNA6 3.7 kbpdeletion-EcoRV fragment this work of pNA1

Preparation of cell extracts. A 500 ml fresh over-night culture of L.lactis or E. coli was harvested by centrifugation at 8 000×g, washedtwice in ice-cold lysis buffer (50 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 25mM NaCl, 7 mM mercaptoethanol). Cell were suspended in 10 ml cold lysisbuffer and disrubted using a French press (Aminco, USA) at 1 000 psi(6,9 MPa). The crude extract was centrifugated for 30 min at 40 000×gbefore glycerol was added to a final concentration of 20%. Aliquots werestored at −20° C. Small scale preparation of E. coli extract was carriedout by centrifugation of a 10 ml overnight culture at 15 000×g, washingtwice with lysis buffer. The cells were resuspended in 0.5 ml lysisbuffer before sonical disruption. After centrifugation at 15 000×g thesupernatant was stored at −20° C. in 20% glycerol.

Determination of endonuclease activity. Type II endonuclease activity invitro was determined by incubating cell extract or purified enzyme withpBluescriptIISK+ or phage λ DNA in 50 mM Tris-HCl pH 7.6, 80 mM NaCl, 10mM MgCl₂, 0.001 M DTT. After 2 hrs at 37° C. the reaction mixtures wereanalyzed by horizontal electrophoresis in agarose gel with TEA buffer(37). In vitro endonuclease activity was determined as an average ofthree independent determinations of the efficiency of plating (EOP)performed as described earlier (23).

Determination of methylase activity. Phage jj50, or λ b2 was purifiedthree times over single plaques and propagated on the selected strains.Phage DNA was isolated by standard procedure (37). Phage DNA wasincubated with purified LlaAI endonuclease (33) as described above. Invitro methylase activity was observed if the DNA was protected againstcleavage with the purified LlaAI endonuclease. In vivo LlaAI methylaseactivity was determined by plaque assays as described previously (23).

Transformation. Protoplast transformation of Lactococcus was conductedas previously described (23). L. lactis MG1614 was grown with 4% glycineand transformed by electroporation using a Bio-Rad Gene Pulser apparatus(Bio-Rad Laboratories, Richmond, Calif., USA) as described by Holo andNes (19). E. coli was transformed by the CaCl₂ standard procedure asdescribed by Sambrook et al (37).

DNA isolation and cloning. Plasmid DNA was extracted from L. Lacks bythe method of Andresen et al (1) and further purified by CsCl-EtBrgradients (37). Plasmid was isolated from E. coli by alkaline lysis (37)and further purified by QIAGEN kit (QIAGEN Inc., Chatsworth, USA) or byCsCl-EtBr gradient (37). Phage DNA was purified by the methods describedby Sambrook et al (37). Deletions were performed with the ‘Erase-a-base’system (Promega Corp., Madison, USA). Blunt ends were created by fillingin with Klenow fragment of DNA polymerase (Boehringer Mannheim,Mannheim, Germany). Restriction endonuclease, T4 ligase, and CalfIntestine Alkaline Phosphase (CIP), were purchased from BoehringerMannheim (Mannheim, Germany) or New England Biolabs (Beverly, USA). Allenzymes and kits were used according to the manufacturersrecommendations.

DNA sequencing. The nucleotide sequence was determined by thedideoxynucleotide chain termination method (39) using double-strandedDNA as template. Restriction fragments and deleted fragments inpBluescriptIISK+ were sequenced in both directions with the Sequenaseversion 2.0 Kit (United States Biochemical, Cleveland, Ohio) using[³⁵S]-dATP (Amersham, England) and standard primers (Stratagene). A fewnon-overlapping sequences were sequenced directly on the intact pFW094plasmid using synthetic primers purchaced from P. Hobolt (Department ofMicrobiology, The Technical University, Denmark). Compressed DNAsequences were resolved by using AmpliCycle™ Sequencing kit (PerkinElmer). Computer analysis was based on GCG sequence analysis program(Version 7.0) (10).

Sequences comparison. The sequence of the LlaAI gene was compared withR-M genes from the GenBank and the EMBL data bank.

Results

Identification and isolation of the R-M encoding plasmid pFW094. Totalplasmid DNA from L. Lactis W9 was isolated and protoplast transformedtogether with pVS2 (chloramphenicol marker) into the plasmid-free strainL. Lactis MG1614 (23). Cm^(R) transformants were tested for phageresistance with phage jj50/MG1614. Phage jj50 was propagated and plaqueassayed on L. Lactis MG1614 and on transformants with increased phageresistance (data not shown). By this method two R-M coding plasmids,pFW093 and pFW094, 12.7 kbp and 15.5 kbp, respectively, were identified.The transformants, TW093 and TW094, carrying pFW093 and pFW094,respectively, were isolated after curing of pVS2. Transformants TW093and TW094 restricted phage jj50 with an EOP of 10⁻³ and 10⁻⁵respectively, indicating that both plasmids code for phage resistancemechanisms.

Cell extracts from L. Lactis W9, TW093 and TW094 were screened for typeII endonuclease activity. Only L. Lactis W9 and the transformant TW094expressed type II activity, designated LlaAI. The endonuclease waspurified and its recognition sequence determined as earlier described(34). The LlaAI system recognized the sequence 5′-↓GATC-3′ (34). LlaAIis an isoschizomer of MboI from Moraxella bovis and DpnII fromStreptococcus pneumoniae. λ DNA and plasmid DNA isolated from dam⁺ E.coli strains were refractory to digestion with the restrictionendonuclease LlaAI, indicating that methylation of the adenine in therecognition sequence protected against cleavage by the LlaAIendonuclease.

Plasmid pFW094 was stably maintained during at least 500 generations inL. lactis MG1614 without any loss of the capability to restrict phages.

Cloning of the R-M system. A restriction map of pFW094 was constructedas shown in FIG. 1. Different restriction fragments were ligated intothe shuttle vector pSA3 and transformed into L. lactis MG1614 selectingfor Ery^(R). Transformants were examined for expression of R-M activity,and the resulting EPOs of phages jj50 and c2 are shown in Table 4.

TABLE 4 Restriction activity of Lactococcus lactis MG1614 harbouringdifferent plasmids on the phages jj50 and c2. EOP of phage: jj50/MGjj50/MG1614[pFW Strain: 1614 c2/MG1614 094] MG1614 1 1 1 MG1614 [pFW094]10⁻⁵ 10⁻⁴ 1 MG1614 [pSNA1] 10⁻⁷ 10⁻⁶ 1 MG1614 [pSNA2] 10⁻¹ 10⁻¹ 1 MG1614[pSNA6] 10⁻⁷ 10⁻⁶ 1 [ ] denotes the plasmid

Plasmid pSNA1 was constructed by cloning a 6,0 kbp EcoRV fragment frompFW094 into pSA3. L. lactis MG1614 [pSNA1] restricted phages jj50 and c2to an EOP of 10⁻⁷ and 10⁻⁶, respectively (Table 4), showing that boththe endonuclease and methylase activity was expressed by the plasmid inL. lactis MG1614 (FIG. 1). Deletions of the HaeIII-EcoRV₂ fragment frompSNA1, resulted in plasmid pSNA2, which did not restrict phages in L.lactis MG1614 (EOP increased from 10⁻⁷ resp. 10⁻⁶ to 10⁻¹), but it hadmethylase activity (FIG. 1). This showed that the HaeIII site waslocated within the gene encoding the endonuclease LlaAI. To localize theLlaAI methylase, the subclones, pSNA3, pSNA4 and pSNA5 were constructedby cloning of restriction fragments of pSNA1 into pSA3, as shown in FIG.1. The plasmid pSNA3 contained the 3.1 kbp BglII-Sau3A₁ fragment, pSNA4the 4.6 kbp BglII-BstXI fragment, and pSNA5 the 2.1 kbp Sau3A₂-EcoRV₂fragment, all inserted into pSA3. None of the three clones restrictedphages and only pSNA4 expressed methylase activity. This indicates thatthe the Sau3A-BstXI fragment is part of the methylase gene.

Plasmid pNA1 was constructed by cloning the 6.0 kbp EcoRV fragment inpBluescriptIISK+ in E.coli XL1-Blue. Deletions were made from the EcoRV₁site of plasmid pNA1.

From one of the generated derivatives pNA6, the overhangs of a 3.7 kbpBssHII-EcoRV₂ fragment was filled in and cloned into the EcoRV site ofpSA3 yielding pSNA6. L. Iactis MG1614 [pSNA6] restricted phage jj50 withan EOP of 10⁻⁷ (Table 2), indicating that the 3.7 kbp BssHII-EcoRV₂fragment is the smallest fragment obtained that contains all theinformation necessary for the expression of the R-M system inLactococcus.

E. coli TC1685 (dam⁻) was transformed with plasmids pSNA1 and pSNA6.None of the transformants expressed endonuclease activity as determinedby plaque assay with phage λ b2 or by assaying E. coli cell extracts forendonuclease activity (data not shown). However, when phage λ b2 DNA waspropagated through TC1685 containing pSNA1 or pSNA6, the λ DNA wasrefractory to digestion with the purified endonuclease LlaAI, indicatingthat the phage DNA was fully methylated and that plasmid pSNA1 and pSNA6had expressed methylase activity.

Deposit. Lactococcus lactis MG1614 transformed with plasmid pSNA1comprising the LlaAI R-M system has been deposited at the BelgianCoordinated Collections of Micro-organisms (BCCM), Laboratorium voorMicrobiologie—Bacteriënverzameling (LMG), Universiteit Gent, Belgium,under the accession number LMG P-15720.

Gene organization and DNA sequence. Subfragments of the 6.0 kbp EcoRVfragment were cloned in the cloning vector pBluescriptIISK+ andtransformed into E.coli XL1-Blue. Several of the clones were deleted inone direction as described in Material and Methods. The nucleotidesequence of a 3.7 kbp region containing the LlaAI R-M genes wasdetermined on both strands. Three open reading frames, ORF1, ORF2 andORF3, were located on the same strand of DNA (SEQ ID No. 1). The ORF1was located between nt 769 to 1620. The ORF1 contained an initiationcodon (ATG) and a putative but unusual ribosomal binding site (RBS)(GGTATAA) located at position 755 to 761. If the initiation codon atposition 769 is used, ORF1 should encode a protein of 284 amino acid.Since the plasmid pSNA4, containing ORF1 and part of ORF2, expressedLlaAI methylase activity (FIG. 1), ORF1 must encode a methylase,M.LlaAIA. The ORF2 is presumably positioned 29 bp downstream of ORF1 andcorrespond to position 1649 to 2419. The initiation codon at position1649 is preceded by a RBS (GGAGG) that conforms well to the consensusRBS (GGAGG) positioned 7 bp upstream of a putative ATG start codon. Ifthis start codon is used, the ORF2 should encode a protein of 257 aminoacids. However, there is another initiation codon at position 1613, butit is not preceded by a consensus RBS. If this is used as the startcodon, the ORF2 should encode a protein of 269 amino acids. It is notyet known which of the two initiation codons is actually used. The ORF3is located between nucleotides 2412 and 3323 with a 8 bp overlap to theORF2 and is preceded by a possible RBS 7 bp upstream (AAGGAG). Theprotein which could be translated from ORF3 starting at position 2412would contain 304 amino acids. Deletions of the region downstream to theHaeIII₁ site at position 3203 abolished endonuclease activity in plasmidpSNA2, showing that ORF3 code for the endonuclease, R,LlaAI. A putativepromoter for the LlaAI R-M system could be located from position 699 to726. The putative promoter region might contain a −10 region (TATTTA),which has good homology to the consensus, and a −35 region (TTAAGA) withlow homology to consensus, 16 bp upstream. Downstream the IIaAI genewith a 6 bp overlap is a putative rho-independent transcriptionalterminator structure with a −G=−26 kJ/mol. No terminator-like structureswere found downstream of ORF1 and ORF2. This indicated that the LlaAIR-M system consisted of three ORFs, possibly transcribed as a singlepolycistronic mRNA. ORF1 codes for a methylase, M.LlaAIA, and ORF3 for arestriction endonuclease, R,LlaAI.

Comparison of amino acid sequences. No primary sequence homology wasfound between the restriction endonuclease R,LlaAI and the methylaseM.LlaAIA or the deduced protein encoded by ORF2, and no sequencesimilarities besides motifs I and II (se below) were found between themethylase M.LlaAIA and the deduced ORF2 encoded protein. The deducedprotein encoded by ORF1 contained the motif I, PFXGXGAhXXG, and themotif II, DhVhXDPPYh, often found in adenine methylases (29) indicatingthat this ORF most likely codes for an adenine methylase. The samemotifs were found in the deduced protein from ORF2 indicating that thisORF most likely also encode an adenine methylase. The deduced amino acidsequences from the LlaAI R-M system were compared to the isoschizomericsystems, DpnII from Diplococcus pneumoniae (28) and MboI from Moraxellabovis (47). Based on amino acid sequence alignments of the three R-Msystems, LlaAI, DpnII and MboI, the calculated identity is shown inTable 5.

TABLE 5 Amino acid sequence identity between the LlaAI R-M system andthe DpnII and MboI R-M systems. DpnM DpnA DpnB MboA MboI MboC M. LlaAIA75% — — 45% — — M. LlaAIB — 86% — — — 50% R. LlaAI — — 32% — 36% —

The three R-M systems are very homologous, especially the two methylasesfrom the DpnII and the deduced M.LlaAIA and ORF2 encoded protein of theLlaAI systems were highly homologous (Table 5). The identity between themethylase M.LlaAIA and DpnM was 75%, and between the methylase DpnA andthe protein from ORF2 86%. The results are consistent with the proposalthat both ORF1 and ORF2 encodes adenine methylases designated M.LlaAIAand M.LlaAIB, respectively, and similar to DpnM and DpnA. Less aminoacid identity was found between the methylases from the LlaAI and theMboI systems, 45% and 50% identity, respectively, (Table 5). Theidentity between the M. LlaAIA and the Dam methylase from E. coli waseven lower (32%, data not shown). As expected the identity between theendonuclease from the three R-M systems was not as significant asbetween the methylases. However, the identity between R,LlaAI, andR,MboI and R,DpnII was 36% and 32%, respectively (Table 5). The LlaAIendonuclease did not show any strong homology to other endonucleasesfrom the GenBank database. The LlaAI and the DpnII systems have the samegene organization: Genes encoding methylases are located upstream of thegene encoding the endonuclease, whereas in the MboI system, the geneencoding the endonuclease is located between the two methylases.

Discussion

LlaAI is the first R-M system from Lactococcus lactis, with knownrecognition sequence, which has been cloned and completely sequenced.The LlaAI system has a recognition sequence 5′-↓GATC-3′ deviating fromthe recognition sequences of ScrFI and LlaAI , recognizing 5′-CC↓NGG-3′and 5′-CC↓WGG-3′, respectively (27), in being more AT rich. This mayhave practical implication, since Lactococcus has AT rich DNA (34-40% CG(40)). The genes were localized to a 3.7 kbp fragment. Interestingly,when the 3.7 kbp fragment cloned in pSA3 (pSNA6) was introduced into L.lactis MG1614, it restricted phage jj50 with an even higher efficiency(10⁻⁷) than the wild-type plasmid. This may be due to a slightly higherplasmid copy number of pSNA6 compared to pFW094 and therefor a higherlevel of expression of the LlaAI genes involved in the R-M phageresistance mechanism. In E. coli TC1685, however, pSNA6 only expressedmethylase activity. Whether this is due to instability of the mRNA or toa regulatory mechanism of the LlaAI endonuclease, that does not functionin E. coli, is not known.

We found that the R-M system consisted of three ORF's putativelytranscribed on a polycistronic mRNA. The gene organization: twomethylase genes followed by the endonuclease gene, is the same as forthe genes encoding the DpnII R-M system (28) but not like the MboIsystem (47), which has the endonuclease gene surrounded by the twomethylase genes.

The two LlaAI methylases showed a very high degree of identity (75 and86%) to the two methylases from the DpnII R-M systems. From the DpnIIR-M system it has been found, that one of the DpnII methylases, DpnM, isan N6-adenine methylase that methylate hemimethylated DNA (9) in thesequence 5′-GATC-3′, whereas the other methylase, DpnA, methylate singlestranded DNA (4). The sensitivity of the LlaAI endonuclease to dammethylation and the high identity between the DpnM and M.LlaAIA suggeststhat M.LlaAIA also is a N6-adenine methylase methylating hemi-methylatedDNA. The high identity between the DpnA and M.LlaAIB suggests thatM.LlaAIB may act like DpnA and methylate single stranded DNA. Thehomologies found between Dam, DpnM, MboA and M,LlaAI indicate a strongrelationship between the four 5′-GATC-3′ N6-Adenine methylases. Despitethe fact, that the Dam methylase has an other biological function thanDpnM, M.MboA and M,LlaAI, it appears that the four methylases originatefrom a common ancestor and that the methylases have diverged intodifferent biological functions. No homology besides the two motifs I andII was found between the two LlaAI methylases, M.LlaAIA and M.LlaAIB,indicating that the two LlaAI methylases are not a result of geneduplication, but have evolved independently of each other.

The homology between the endonuclease from LlaAI and those from DpnII(32%) and MboI (36%) is unusually high for isoschizomers, indicating acommon ancestor. Similar results have been seen before (52), but mostoften no similarities are seen between isoschizomers (52).

EXAMPLE 2

Here we report the cloning and nucleotide sequence of the genes codingfor the LlaBI system from Lactococcus lactis subsp. cremoris W56. TheLlaBI endonuclease is an isoschizomer to SfcI from Enterococcus faecium.L. Iactis W56 has previously been isolated from the Danish mixed Cheddarstarter, TK5 (22). It was shown that L. lactis W56 harbors at leastthree plasmids, pJW563, pJW565, and pJW566, which encode distinct R-Msystems (23). It was found that transformants harbouring the plasmidpJW563 expressed type II activity, named LlaBI (34).

Materials and Methods

Strains, phages, plasmids and growth conditions. The strains Lactococcuslactis subsp. cremoris W56, obtained from E. Waagner Nielsen (22) andLactococcus lactis subsp. cremoris MG1614, originally classified asLactococcus lactis subsp. lactis (12; 14), obtained from Atte vonWright, were grown at 30° C. in M17 media (Oxoid) supplemented with 0,5%glucose (GM 17) and 5 mM CaCl₂ when phages were used. The antibioticswere purchased from Sigma and were used at the following concentrations:chloramphenicol, 10 μg/ml; erythromycin and tetracycline, 5 μg/ml.Eschericia coli strains XL1l-Blue (Stratagene) and HB101 (3) were grownat 37° C. in LB supplemented with chloramphenicol, erythromycin,tetracycline or ampicillin at 10, 100, 12,5 and 100 μg/ml, respectively,when needed. The isometric headed phage jj50 and the prolate headedphage c2 (25) were propagated and titrated by the method of Terzaghi andSandine (34). λ b2 phage was propagated as described by Sambrook (37).The plasmids used in this study are shown in Table 6.

TABLE 6 Plasmids. Plasmid Relevant characteristics Source or referencepJW563 r⁴/m⁴ (23) pSA3 shuttle vector (7) pBluescriptIISK+ StratagenepUC7, erm pUC7::1.1 kbp HinPI pIL253 W. M. de Vos, erm NIZO, Ede, TheNetherlands pJWC1 pJW563::cam cassette in ClaI this work site pJWC2pJWC1::deletion of 1.2 kbp this work BCII fragment pJWE1 pJWC1::1.1 kbpery cassette in this work BgIII site pSNB1 pSA3::4.0 kbp HindIII pJW563this work pSK-cm1 pBluescriptIISK+::3.9 kbp pVC5 Finn K. Vogensen campAG55 pSK-cm1::3.1 kbp HindIII this work pJW565;::6.4 kbp EcoRI pJW563

Preparation of cell extracts. A 500 ml fresh over-night culture of L.lactis was harvested by centrifugation at 8 000×g washed twice in 10 mlcold lysis buffer (50 mM Tris-HCl pH 7.6, 10 MM MgCl₂, 25 mM NaCl, 7 mMmercaptoethanol) and suspended in 10 ml cold lysis buffer. A Frenchpress (Aminco, USA) was used to disrupt the cells at 1 000 psi (6,9MPa). The crude extracts were centrifugated for 30 mn at 40 000×g beforeglycerol was added to the supernatant to a final concentration of 20%.Aliquots of the extract were stored at −20° C.

Determination of endonuclease activity. Type II endonuclease activity invitro was determined by incubating cell extract or purified enzyme (34)with pBluescriptIISK+ or phage λ DNA in 50 mM Tris-HCl pH 7.9, 10 mMNaCl, 10 mM MgCl₂, 100 μg/ml BSA, 0.001 M DTT. After 2 hrs incubation at37° C. the reaction mixture was analyzed by horizontal electrophoresisin agarose gel with TAE buffer (37). The in vivo endonuclease activitywas determined as an average of three independent determinations of theefficiency of plating (EOP) performed as described earlier (23).

Transformation. L. Iactis MG1614 was grown with 4% glycine andtransformed by electroporation using a Bio-Rad Gene Pulser apparatus(Bio-Rad Laboratories, Richmond, USA) as described by Holo and Nes (19).E. coli strains were transformed by the CaCl₂ standard procedure (37).

DNA isolation and cloning. Plasmid DNA was extracted from L. lactisstrains by the method of Andresen et al (1) and purified by the CsClgradient method (37). Plasmid DNA isolation from E. coli was performedby alkaline lysis (37). DNA was further purified by QIAGEN coloums(QIAGEN Inc., Chatsworth, USA) or by CsCl-EtBr gradients (37). Phage DNAwas isolated as described by Sambrook et al. (37). Restrictionendonucleases, T4 ligase, and Calf Intestine Alkaline Phosphase (CIP)were purchased from Boehringer Mannheim (Mannheim, Germany) or NewEngland Biolabs (Beverly, USA). Deletions of subcloned fragments wereobtained by using the ‘Erase-a-base’ system (Promega Corp., Madison,USA). All enzymes and kits were used according to the manufacturer'srecommendations.

DNA sequencing. The nucleotide sequence was determined by thedideoxynucleotide chain termination method (39) using double-strandedDNA as templates. Restriction fragments and deleted fragments cloned inpBluescriptIISK+ were sequenced using the Sequenase version 2.0 kit(United States Biochemical, Cleveland, Ohio, USA). The sequencing wasdone in both directions using [³⁵S]-dATP (Amersham, England) andstandard primers (Stratagene) complementary to the region of the plasmidupstream of the deleted fragments. A few regions were sequenced by PCRdirectly from the intact pJW563 plasmid using synthetic primers providedby P. Hobolt (Department of Microbiology, The Technical University,Denmark). In case of compression, DNA sequencing was carried out withAmpliCycle™ Sequencing kit (Perkin Elmer). All computer analysis wasdone with GCG sequence analysis program (Version 7.0) (10).

Sequence comparisons. The sequence of the LlaBI genes was compared toR-M genes from the GenBank and the EMBL data bank.

Results

Cloning and localization of the LlaBI R-M system. Cell extracts from L.lactis W56, T1.1 [pJW563], T21.5 [pJW565] and T46.22 [pJW566] werescreened for type II endonuclease activity. Only L. lactis W56 and thetransformant T1.1 expressed type II activity, designated LlaBI. Arestriction map of the plasmid pJW563 was made (FIG. 2). Direct cloningof the entire R-M system in E. coli in different vectors was notsuccessful (data not shown). Therefor cloning of pJW563 was carried outin L. lactis MG1614. Due to problems in cloning the entire system in L.lactis MG1614 it was decided to determine the location of the genes forthe R-M system on the plasmid pJW563. A chloramphenicol resistancecassette was inserted into one of the ClaI sites, resulting in theplasmid pJWC1 (data not shown). L. lactis MG1614[pJWC1] expressed R-Mactivity like the wild type plasmid. Bidirectional deletions were madeshowing that the R-M system was located around the BglII site (data notshown). An erythromycin cassette with compatible BamHI linker ends wasinserted into the unique BglII site of pJW563, and the resultingplasmid, pJWE1, was electroporated into L. lactis MG1614 (FIG. 2). Celllysate of the transformant did not express any LlaBI endonucleaseactivity and the transformant did not restrict phages. However, thetransformant L. lactis MG1614[pJWE1] expressed methylase activity foundby the fact that phages, propagated on the transformant, was notrestricted by transformants containing pJW563. Deletion of the 1.2 kbpBclI fragment from pJWEI resulted in plasmid pJWE2, which showed neitherendonuclease nor methylase activity (FIG. 2). These results indicatedthat the endonuclease gene was located near the BglII site, whereas atleast a part of the methylase gene was located on the 1.2 kbp BclIfragment. The entire R-M system was then cloned in the vector pSA3 on a4.0 kbp HindIII fragment containing the BglII and BclII sites, resultingin the plasmid pSNB1. Crude cell extracts prepared from L. lactisMG1614[pSNB1] transformants expressed LlaBI endonuclease activity (datanot shown) and restricted phage jj50 with an (EOP) of 10⁻⁴ and phage c2with and EOP of 10⁻³. Phage jj50 propagated on L. lactis MG1614[pSNB1]was not restricted by the L. lactis MG1614[pJW563] strain, indicatingthat phage jj50 DNA had been methylated, suggesting that the plasmidpSNB1 carried the genes encoding both the endonuclease and methylasefrom the LlaBI R-M system and that they function as a phage resistancemechanism. It was not possible to transform E. coli HB 101 with theplasmid pSNB1, which harboured the HindIII-fragment cloned in pSA3,indicating that the entire LlaBI R-M system is lethal to E. coli. TheLlaBI encoding genes were also cloned as a 6.4 kbp EcoRI fragment frompJW563 in a BluescriptIISK+ derivative carrying a cassette encodingchloramphenicol resistance and a replicon from pJW565, resulting inplasmid pAG55. L. lactis MG1614[pAG55] restricted phage jj50 at the sameorder of magnitude as the wild-type plasmid pJW563.

Deposit. Lactococcus lactis subsp. lactis MG1614 transformed withplasmid pAG55 comprising the LlaBI R-M system has been deposited at theBelgian Coordinated Collections of Microorganisms (BCCM), Laboratoriumvoor Microbiologie—Bacteriënverzameling (LMG), Universiteit Gent,Belgium, under the accession number LMG P-15719.

Gene organization and DNA sequence. It was not possible to clone the 4.0kbp HindIII fragment in pBluescriptIISK+ in E. coli XL1-Blue, indicatingagain that the endonuclease expression may be lethal to E. coli. Severalof the clones were deleted in one direction as described in Materialsand Methods. The nucleotide sequence of the 4.0 kbp HindIII fragmentcontaining the LlaBI R-M genes was determined on both strands. Two majorORFs, ORF1 and ORF2, were found in the sequence (SEQ ID No. 9): ORF1comprised 1740 bp, with a coding potential for a 580 aa protein, andORF2 comprised 897 bp, capable of coding for a 299 aa protein. The twoORFs were separated by 302 bp and transcribed divergently. Both ORFswere preceded by putative Shine-Dalgarno sequences 7 bp in front of theATG start codon. Putative −10 (sequence TATAAT and TATAAG) and −35(sequence TTGACT and TCGTAA) consensus regions were found upstream ofboth ORFs. The sequenced region had only one BglII site, and it waslocated 138 bp downstream of the putative start codon in ORF2. ThisBglII site was inactivated in plasmids pJWE1 and pJWC1, which did notexpress endonuclease activity, showing that ORF2 codes for the LlaBIendonuclease. The 1.2 kbp BclI fragment, which was deleted in plasmidpJWE2 (FIG. 2), was found at position 1305 to 2520 covering 856 bpdownstream in ORF1, showing that ORF1 codes for the methylase, M,LlaBI.A secondary structure indicating a putative terminator loop was founddownstream of the methylase gene with a 2 bp overlap. The results showthat the LlaBI R-M system consists of a methylase, M-LlaBI, putative ofa 580 aa protein (65 kDa) and an endonuclease, R.LlaBI, putative of a299 aa protein (33 kDa), transcribed divergently.

The r.llaBI gene is preceded by one short ORF, which extends over 90 bpand may code for a small protein of 30 amino acids. This ORF is alignedin the same orientation as the r.IlaBI gene and separated therefrom by110 bp.

Comparison of amino acid sequences. No primary sequence similaritieswere found between the restriction endonuclease R.LlaBI and thecorresponding methylase M.LlaBI, or with other type II restrictionendonucleases. The deduced amino acid sequence of the M.LlaBI methylasewas compared with the amino acid sequence of other methylases in thedata banks. The motif II, DhVhXDPPYh, which is common to all knownadenine and cytosine methylases (29) was found in the sequence fromnucleotide 1717 to 1688. The sequence lacked the motifs common tocytosine methylases (30). A motif similar to motif III (21), which isassociated with adenine methylases like Eco57 1, PstI, PaeR71 and BsuBI,recognizing the sequence CTxxAG, was also found in LlaBI. Since thesecomparisons of the amino acid sequence of the M.LlaBI methylase to othertype II methylases revealed a significant similarity to N6-adeninemethylases and a lack of the numerous conserved motifs common tocytosine methylases, the M.LlaBI methylase most likely is a N6-adeninemethylase.

Discussion

The R-M coding plasmid, pJW563, isolated from L. lactis W56, waspreviously reported to restrict the isometric headed phage jj50 with anefficiency of plating (EOP) of 10⁻³ and the prolate headed phage with anEOP of 10⁻², clearly showing that it encodes a phage resistancemechanism (23). The system exhibiting type II endonuclease activity,designated R.LlaBI, was purified and its recognition sequence determinedto be 5′-C↓TRYAG-3′ (34). Two plasmids, pSNB1 and pAG55, harbouring thegenes encoding the LlaBI R-M system, have been constructed. In L. lactisMG1614 the plasmids pSNB1 and pAG55 had the ability to restrictlactococcal phages with an EOP at the same level as found for thewild-type plasmid, pJW563. This showed that the genes can be cloned andused to increase the phage resistance in Lactococcus strains.

By cloning and sequencing the LlaBI R-M system, it was found that theR-M system consists of two ORFs putatively transcribed divergently. Themethylase is encoded by an ORF of 1740 bp capable to code for a proteinof 580 amino acid, while the putative endonuclease is encoded by an ORFof 897 bp capable to code for a protein of 299 amino acids. The size ofthe methylase (65 kDa) is considerably larger than most of the sequencedmethylases, indicating that the M.LlaBI methylase may be a monomer. Thededuced size of the restriction endonuclease (33 kDa) is comparable tothe sizes of other endonucleases (51). Probably the endonucleasefunctions as a dimer like many other type II endonucleases. The missingprimary sequence similarities between the M.LlaBI and the correspondingR.LlaBI supports the general assumption that type II restrictionendonucleases and methylases are evolutionary unrelated and interactwith target DNA sequences by different mechanisms (49).

Preceding the r.llaBI gene was a small ORF (90 bp) potentially encodinga protein of 30 amino acids. Probably this protein is too small to actas a trans-acting positive regulator of the r.llaBI gene, similar topvuIIC, found in the PvuII system (43) and other systems withdivergently transcribed genes.

The motif II is presumably involved in the general steps of DNAmethylation, probably in the transfer of the methyl group (29). Thestructural similarity of the methylases recognizing the sequence CTXXAGsuggests that motif III may be involved in the sequence recognition ofthe methylases. From cytosine methylases, however, experimental evidencesuggests that the large amount of conserved motifs (29) may be involvedin the proper folding of the protein, while the variable regions may beresponsible for sequence specificity (35). It cannot be excluded thatmotif III is involved in the folding of the methylases.

During the cloning of the LlaBI system it was found that the plasmidpJW563 was resistant to digestion by the PstI restriction endonuclease(data not shown) although subclones of pJW563 containing fragments ofthe M.LlaBI methylase were not resistant to PstI restriction. The PstIendonuclease recognises 5′-CTGCA↓G-3′ and cuts as indicated by thearrow. LlaBI can recognise the same sequence, 5′-C↓TGCAG-3′ (and5′-C↓TATAG-3′), but will cut the recognition sequence at a differentplace (LlaBI generates 5′-overhangs while PstI gives 3′-overhangs). Thisindicates, that the adenine in the PstI recognition sequence has beenmethylated by the M.LlaBI methylase. This result, together with thehomology found between the M.LlaBI methylase and other adeninemethylases, and the lack of homology to common motifs in cytosinemethylases, indicate that the M.LlaBI methylase is a N6-adeninemethylase.

The average G+C content of the LlaBI genes is 27,8% (31,5% for ther.llaBI and 25,7% for m.llaBI), which is much lower than the 34 to 43%G+C content normally found in lactococci by measuring the meltingtemperature (40). This may indicate that the LlaBI R-M system originatesfrom genus other than Lactococcus.

EXAMPLE 3

L. lactis W39 has previously been isolated from the Danish mixed Cheddarstarter, TK5 (33). We found, as shown in Table 2, that L. lactis W39expressed type II endonuclease activity, which we designated LlaDI. Herewe report the cloning and nucleotide sequence of the genes coding foranother type II R-M system from L. lactis W39, designated LlaDII, withan endonuclease having a different restriction pattern from that ofLlaDI.

Materials and Methods

Strains, phages, plasmids and growth conditions. The strains andbacteriophages used in this study are listed in Table 7. The L. lactisstrains were grown at 30° C. in M17 media (Oxoid) supplemented with 0,5%glucose (GM17) and 5 mM CaCl₂ when phages were used. Escherichia coli(E. coli) strains were grown at 37° C. in LB. The antibiotics (Sigma)were used at the following concentrations: in L. lactis:chloramphenicol, 6 μg/ml; in E. coli: chloramphenicol, 20 μg/ml;tetracycline, 12,5 μg/ml; and ampicillin, 100 μg/ml. Lactococcal phagepropagation and plaque assays were carryed out as described by Terzaghiand Sandine (44). The plasmids used in this study are shown in Table 8.

TABLE 7 Bacterial strains and bacteriophages used Bacterial strain orphage Relevant characteristics Reference or source L. lactis subsp.cremoris: W39 industrial strain, multiple E. Waagner Nielsen (22)plasmids MG1614 plasmid-free, host for jj50, A. von Wright (12) p2 andc2 phages; transfor- mation host. LM2301 plasmid-free, host for jj50,Stephen Wessels (48) p2 and c2 phages; transfor- mation host. E. coli:XL1-Blue MRF Transformation host Strategene Phages: jj50 Small isometricheaded, (23) 936 species p2 Small isometric headed, T. R. Klaenhammer936 species c2 Prolate headed, c2 species T. R. Klaenhammer (25)

TABLE 8 Plasmids used in this study. Reference Plasmid Relevantcharacteristics or source pHW393 r+/m+ this work pCI3340 shuttle vector(15) pVS2 shuttle vector (53) pBluescript II Stratagene SK+ pSA3 plasmidDao and Ferretti (7) pCAD1 pCI3340::2.4 kb PstI-EcoRI this work pCAD2pCI3340::5.4 kb XbaI-EcoRI this work pSAD1 pBluescriptIISK+::0.9 kbPstI-XhoI this work pSAD2 pBluescriptIISK+::1.5 kb this work XhoI-EcoRI

Preparation of cell extracts. A 1 l fresh over-night culture of L.lactis was harvested and washed once in 10 ml cold lysis buffer (50 mMTris-HCl pH 7.6, 10 mM MgCl₂, 25 mM NaCl, 7 mM mercaptoethanol) andsuspended in 12-15 ml cold lysis buffer. A French press (Aminco, USA)was used to disrupt the cells at 1500 psi. The crude extracts werecentrifugated for 2 hrs at 180 000×g before glycerol was added to thesupernatant to a final concentration of 20%. Aliquots of the extractwere stored at −20° C.

Determination of endonuclease activity. Type II endonuclease activity invitro was determined by incubating cell extract or partially purifiedenzyme with plasmid DNA in NEBuffer 2 (10 mM Bis-Tris-propane-HCl pH7.0, 10 mM MgCl₂, 1 mM DTT) from Biolabs (New England, USA). After 1 hrincubation at 37° C., the reaction mixture was analyzed byelectrophoresis in agarose gel with TAE buffer (37). The in vivoendonuclease activity was determined as an average of three independentdetermination of the efficiency of plating (EOP) performed as describedearlier (23).

Purification of restriction endonucleases. Cell extract, made asdescribed, was purified by an one-step FPLC chromatographic procedureusing a Mono Q column in buffer A (50 mM Tris-HCl pH 7.6, 10 mM MgCl₂, 5mM mercaptoethanol). The enzyme was eluted with 1 M KCl in buffer A andcollected in small fractions, which were assayed for endonucleaseactivity.

Determination of methylase activity. Plasmid DNA was incubated withpurified LlaDII endonuclease as described above. Methylase activity wasobserved if the plasmid DNA encoding the methylase was protected againstcleavage with the purified LlaDII endonuclease. In vivo LlaDII methylaseactivity was determined by plaque assays.

Transformation. L. Lactis LM2301 was grown with 3% glycine andtransformed by electroporation using a Bio-Rad Gene Pulser apparatus(Bio-Rad Laboratories, Richmond, USA) as described by Holo and Nes (19).E. coli strains were transformed by the CaCl₂ standard procedure (37).

DNA isolation and cloning. Plasmid DNA was extracted from L. Iactisstrains by the method of Andresen et al (1) and purified by the CsClgradient method (37). Plasmid DNA isolation from E. coli was performedby alkaline lysis and CsCl gradient method (37) or by QIAGEN columns(QIAGEN Inc., Chatsworth, USA). Restriction endonucleases, T4 ligase,and Calf Intestine Alkaline Phosphase (CIP) were purchased fromBoehringer Mannheim (Mannheim, Germany), Amersham (Buckinghamshire, UK)or New England Biolabs (Beverly, USA). All enzymes and kits were usedaccording to the manufacturers recommendations.

DNA sequencing. Double-stranded DNA templates for sequencing wereobtained by subcloning various DNA fragments in pBluescript II SK+.Deletions of the entire fragments were obtained by using the‘Erase-a-base’ system (Promega Corp., Madison, USA). The nucleotidesequence was determined by standard dideoxy sequencing using Auto Read™Sequencing kit (Hoefer Pharmacia Biotech Inc., San francisco, USA). Thefragments were sequenced on both strands using universal and reverseprimers (Stratagene). Computer analyses were performed with the GCGsequence analysis program (Version 8.0) (10).

Sequence comparisons. The sequence of the LlaDII genes was compared withR-M genes from the NCBI database.

Results

Identification of a plasmid encoding the LlaDII R-M system. Totalplasmid DNA from L. lactis W39 was cotransformed with pVS2 into L.lactis MG1614 selecting for chloramphenicol resistance (23). The phagesensitivity of the transformants were determined by crossstriking withphage jj50. It was found that cell extract from transformant L. lactis39.26, which harboured plasmid pHW393 besides pVS2, expressed type IIendonuclease activity designated LlaDI. L. lactis 39.26 restricted thesmall isometric headed phages jj50 and p2 with an efficiency of plating(EOP) of 10⁻⁴ and the prolate headed phage c2 with an EOP of 10⁻².Phages propagated on the transformant circumvented the restriction,showing that plasmid pHW393 encodes a R-M system. The transformant 39.26was cured for plasmid pVS2 by treatment with novobiocin, and pHW393 wasretransformed into L. lactis LM2301. This transformant T39.3 restrictedthe phages jj50, p2 and c2 with the same EOP as tranformant 39.26.

Cloning and localization of the LlaDII R-M system. A restriction map ofthe plasmid pHW393 was made (FIG. 3). Two plasmids, designated pCAD1 andpCAD2, containing a 2.4 kbp PstI-EcoRI fragment and a 5.4 kbp XbaI-EcoRIfragment, respectively, in the shuttle vector pCI3340, were constructedand transformed into L. lactis LM2301. Both transformants restrictedphages jj50 and p2 with an EOP of 10⁻³. Phages propagated ontransformant L. lactis LM2301 [pCAD1] circumvented the restriction,showing that plasmid pCAD1 encodes a R-M system, which was designatedLlaDII. It was only possible to transform E. coli XL 1-Blue MRF′ withplasmid pCAD1; when plasmid pCAD2 was used for transformation onlydeleted plasmids were obtained.

Deposit. Lactococcus lactis subsp. cremoris LM2301 transformed withplasmid pCAD1 comprising the LlaDII R-M system has been deposited at theBelgian Coordinated Collections of Microorganisms (BCCM), Laboratoriumvoor Microbiologie—Bacteriënverzameling (LMG), Universiteit Gent,Belgium, under the accession number LMG P-16901.

Determination of the recognition site. The restriction endonuclease,LlaDII, of the transformant L. lactis LM2301 [pCAD1] was partiallypurified from cell extract. The restriction patterns of λ DNA digestedwith the endonucleases from L. lactis LM2301 [pCAD1] and L. lactis 39.26were different from each other (data not shown). When pBluescript IISK+and pSA3 were digested with the LlaDII and BsoFI restrictionendonucleases identical restriction pattern were obtained (FIG. 4),showing that the restriction endonucleases LlaDII and BsoFI expressedthe same type II activity and are isoschizomers. BsoFI recognized andcleaved the sequence 5′-GC↓NGC-3′ (31).

Gene organization and DNA sequence. The PstI-EcoRI fragment wassubcloned as PstI-XhoI and XhoI-EcoRI fragments in pBluescript II SK+resulting in plasmids pSAD1 and pSAD2, respectively. Deletions were madeas described in Materials and Methods. The nucleotide sequence of the2.4 kb fragment revealed 2 major open reading frames (ORFs) (SEQ ID No.12). ORF1 was putatively 540 bp with a coding potential for a protein of180 amino acids (SEQ ID No. 13), and ORF2 was 951 bp capable of codingfor a protein of 316 amino acids (SEQ ID No. 14). The two ORFs areseparated by 108 bp and arranged tandemly with ORF1 preceding ORF2. ORF2was preceded by a putative Shine-Dalgarno sequence with good identity toconsensus, 8 bp in front of the ATG start codon. Plasmid pSAD2,harbouring only ORF2, was resistent to digestion by the LlaDIIendonuclease, showing that ORF2 had its own promotor and encodes amethyltransferase, which can be expressed in E. coli (data not shown).The results show that the LlaDII R-M system consists of twoconsecutively transcribed genes, where ORF2 carry the gene for amethyltransferase, M.LlaDII.

Comparison of amino acid sequences. The deduced amino acid sequences ofORF1 and ORF2 were compared with the amino acid sequences of othermethylases in the databases. The first ten amino acids encoded by theputative ORF1 may be doubtful as the sequencing first gave base no. 744in SEQ ID No 12 as TT. However, from base no. 773 this reading framegives a high homology with the endonuclease of the Bsp6I R-M system. Thededuced amino acid sequence of ORF2 showed 60% identity and 76%similarity to the methylase from the Bsp6I R-M system (31) and itcontained several amino acid sequence motifs characteristic forC-5-cytosine methyltransferases (50). This also indicates that ORF2codes for a C-5-cytosine methyltransferase.

Discussion

The 8.9 kb naturally occurring plasmid pHW393 was isolated from L.lactis W39. Transformants L. lactis LM2301 [pHW393] and L. lactis LM2301[pCAD1] were both able to restrict phages and this restriction wascircumvented by propagation of surviving phages on the respectivetransformants, showing that plasmid pHW393 and the 2.4 kbp PstI-EcoRIfragment in pCAD1 both code for a restriction/modification system, andthat both plasmids can be used to increase the level of phage defence inLactococcus lactis. The 2.4 kbp PstI-EcoRI fragment cloned in pCI3340had in Lactococcus the ability to restrict lactococcal phages with oneorder of magnitude lower EOP than found for the wild-type plasmid,pHW393, indicating that the expression of the cloned LlaDII R-M systemmay be depending of the plasmid copy number, or it may require someadditional factors or that there are two R-M systems present on plasmidpHW393. Since plasmid pHW393 was found to express LlaDI endonucleaseactivity, and plasmid pCAD1 containing the 2.4 kb PstI-EcoRI fragmentthereof expresses LlaDII activity, plasmid pHW393 must encode two typeII R-M systems. This is the first time that two type II R-M systems havebeen found on the same plasmid.

The 2.4 kbp PstI-EcoRI fragment harbours two tandemly arranged genes,LlaDIIR and LlaDIIM, which encode a restriction endonuclease and aC-5-cytosine methyltransferase, respectively. The R.LlaDII gene precedesthe M.LlaDII gene and they are separated by 108 bp. Since plasmid pSAD2harbouring only the M.LlaDII gene expressed methylase activity in E.coli, these genes are most likely transcribed as two monocistronicmRNAs.

The endonuclease of the LlaDII R-M system is an isoschizomer of BsoFIrecognizing the sequence 5′-GC↓NGC-3′, showing that the LlaDII R-Msystem is a type II system. This is the first time a R-M system, whichrecognizes the sequence 5′-GC↓NGC-3′, has been identified and cloned inLactococcus lactis.

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53. von Wright, A., Tynkkynen, S. and Suominen, M.: Cloning of aStreptococcus lactis subsp. lactis chromosomal fragment associated withthe ability to grow in milk. Appl. Environ. Microbiol. 53 (1987):1584-1588.

14 3695 base pairs nucleic acid double linear DNA (genomic) NO NOLactococcus lactis subsp. cremoris W9 CDS 769..1620 experimental/codon_start= 769 /product= “LlaAI -GATC- N-6-adenine methylase A”/evidence= EXPERIMENTAL /gene= “ORF” /number= 1 /standard_name= “Genecoding for M.LlaAIA” /label= m-llaAIA CDS 1613..2419 experimental/codon_start= 1613 /product= “LlaAI -GATC- adenine methylase B”/evidence= EXPERIMENTAL /gene= “ORF” /number= 2 /standard_name= “Genecoding for M.LlaAIB” /label= m-llaAIB CDS 2412..3323 experimental/codon_start= 2412 /product= “LlaAI restriction endonuclease” /evidence=EXPERIMENTAL /gene= “ORF” /number= 3 /standard_name= “Gene coding forLlaAI restriction endonuclease” /label= r-llaAI 1 ATATAAGATA TATAAATCAGTTCGCCTTTT TCTACTCCGT TCTAAAATCT TAAAATCAAG 60 GTCAAAAGAA AAAGTCAAAACCATTGAATT GAGGTTCTAA AATTAAACTC CCTGCGTTGC 120 TCTTGGCTGC CGCTTGTACACTCTGATTTT ATATTAGATA CATTCTGCCA TTAAAAAGAA 180 CTCCTAACGG TCGTGGCTACTTTGTTTAGT CTAAACGCTT TAAATAGTCC TACAAGCTCA 240 TATTTTGCCT TTTAAGCGATTTTAAACGTG AGTTAGTAAT AATTATCATG GATAAAAGAA 300 AAAGCCCTTA AATAGGCTTGTATGTAATTG ACTAAAACGT ACAATTTAGC TTTTAAATAT 360 GACCCTTATT TATGACCTGCTCTAACCTCA CTATTCATCA GCATTCAAAA AAGAGGTCAA 420 AACTGTTAAG TTATGAGCTGAATAGATTTT ATTAAATTTT ATTTGGTTTA AAAGACCAAT 480 TATCTATTTT TTAACAAACACTAAAATAGA TTTTTTGGAA AACTTTGCAA CAGAACCAGC 540 AATCTGATGT TGCGAGATGGACGTTCTTTC GGTTTTGAAC CTCAAGGGGA ACACTCGTTT 600 GATAAAGCGT CTCAATGGTTGTCAGTAAAC AAACAAAAAC TTTTGGAAGT GTGCTATTAT 660 AAGTCATATA AGTCGTGCGCTTTCTAATGC TTAGTGCTTT AAGATTAGGA TAGCACGACT 720 TATTTATTTT CCAATAAAATTAACTAGCAA TTCGGGTATA ATATATTT ATG AAT TTA 777 Met Asn Leu 1 TTA CAA AAAAAC AAG ATC AAC TTA CGT CCG TTT ACT AAA TGG ACA GGT 825 Leu Gln Lys AsnLys Ile Asn Leu Arg Pro Phe Thr Lys Trp Thr Gly 5 10 15 GGG AAA AGG CAACTA CTG CCA CAC ATT CAA TAC CTA ATG CCA GAA AAA 873 Gly Lys Arg Gln LeuLeu Pro His Ile Gln Tyr Leu Met Pro Glu Lys 20 25 30 35 TAC AAT CAT TTTTTC GAA CCT TTT ATT GGT GGT GGC GCT TTG TTT TTT 921 Tyr Asn His Phe PheGlu Pro Phe Ile Gly Gly Gly Ala Leu Phe Phe 40 45 50 GAA CTC GCT CCT CAAAAA GCA GTT ATT AAC GAC TTC AAT TCT GAG CTT 969 Glu Leu Ala Pro Gln LysAla Val Ile Asn Asp Phe Asn Ser Glu Leu 55 60 65 ATA AAC TGT TAC CGG CAGATG AAA GAT AAT CCT GAG CAA TTG ATA GAA 1017 Ile Asn Cys Tyr Arg Gln MetLys Asp Asn Pro Glu Gln Leu Ile Glu 70 75 80 TTG TTG ACT AAT CAT CAG CGGGAA AAT TCT AAA GAA TAT TAT TTA GAC 1065 Leu Leu Thr Asn His Gln Arg GluAsn Ser Lys Glu Tyr Tyr Leu Asp 85 90 95 TTA CGT TCT TCT GAT AGA GAT GGAAGA ATT GAT AAG ATG AGC GAA GTT 1113 Leu Arg Ser Ser Asp Arg Asp Gly ArgIle Asp Lys Met Ser Glu Val 100 105 110 115 GAA CGT GCT GCT AGA ATT ATGTAT ATG CTA CGT GTT GAT TTT AAT GGT 1161 Glu Arg Ala Ala Arg Ile Met TyrMet Leu Arg Val Asp Phe Asn Gly 120 125 130 TTA TAT CGT GTT AAT TCG AAAAAC CAG TTT AAT GTG CCT TAT GGA AGA 1209 Leu Tyr Arg Val Asn Ser Lys AsnGln Phe Asn Val Pro Tyr Gly Arg 135 140 145 TAT AAA AAT CCT AAG ATA GTTGAT AAA GAA TTG ATT GAA AGT ATT TCC 1257 Tyr Lys Asn Pro Lys Ile Val AspLys Glu Leu Ile Glu Ser Ile Ser 150 155 160 GAG TAC TTG AAT AAC AAT TCTATT AAG ATC ATG AGT GGA GAT TTT GAA 1305 Glu Tyr Leu Asn Asn Asn Ser IleLys Ile Met Ser Gly Asp Phe Glu 165 170 175 AAA GCC GTT AAA GAA GCA CAGGAT GGA GAT TTT GTT TAT TTC GAC CCT 1353 Lys Ala Val Lys Glu Ala Gln AspGly Asp Phe Val Tyr Phe Asp Pro 180 185 190 195 CCA TAC ATT CCA CTT TCTGAA ACT AGC GCC TTT ACT TCT TAT ACA CAC 1401 Pro Tyr Ile Pro Leu Ser GluThr Ser Ala Phe Thr Ser Tyr Thr His 200 205 210 GAA GGC TTT AGC TAC GAAGAT CAA GTT AGG CTA AGA GAT TGT TTC AAA 1449 Glu Gly Phe Ser Tyr Glu AspGln Val Arg Leu Arg Asp Cys Phe Lys 215 220 225 CAG TTA GAT TCA AAA GGGGTA TTC GTC ATG CTT TCA AAT TCT TCA AGC 1497 Gln Leu Asp Ser Lys Gly ValPhe Val Met Leu Ser Asn Ser Ser Ser 230 235 240 CCT TTA GCG GAG GAA TTATAT AAA GAT TTT TAC ATC CAT AAA ATT GAA 1545 Pro Leu Ala Glu Glu Leu TyrLys Asp Phe Tyr Ile His Lys Ile Glu 245 250 255 GCT ACT CGA ACA AAT GGGGCT AAA TCA TCT AGT CGT GGA AAA ATC ACT 1593 Ala Thr Arg Thr Asn Gly AlaLys Ser Ser Ser Arg Gly Lys Ile Thr 260 265 270 275 GAA ATC ATC GTA ACCAAT TAT GGC AAT TAACGAATAT AAGTATGGAG 1640 Glu Ile Ile Val Thr Asn TyrGly Asn 280 GTGTTTTAAT GATAAAACCA TACTATGAAA AAGAAAACGC AATTCTCGTTCACGCAGATT 1700 CATTTAAATT ATTAGAAAAA ATTAAACCTG AAAGCATGGA CATGATATTTGCTGACCCTC 1760 CTTACTTTTT AAGTAATGGA GGAATGTCAA ATTCAGGTGG TCAAATTGTTTCTGTTGATA 1820 AAGGGGATTG GGATAAAATT TCTTCATTTG AAGAAAAACA TGACTTTAATAGACGTTGGA 1880 TTAGGTTAGC AAGATTGGTT TTAAAACCCA ACGGAACTAT TTGGGTTTCCGGAAGCCTTC 1940 ATAACATATA TTCTGTCGGG ATGGCGCTGG AACAGGAAGG TTTCAAAATCTTAAATAATA 2000 TAACTTGGCA AAAGACAAAT CCTGCACCTA ATCTATCATG TCGGTACTTCACCCACTCTA 2060 CAGAGACAAT TTTATGGGCA AGAAAGAACG ATAAAAAATC TCGCCATTATTATAACTATG 2120 AATTGATGAA AGAGTTTAAT GACGGGAAAC AAATGAAAGA TGTTTGGACAGGTAGTCTGA 2180 CAAAAAAATC AGAAAAATGG GCTGGGAAAC ATCCAACTCA GAAGCCAGAGTATATTTTAG 2240 AACGGATAAT CTTAGCTAGT ACAAAGGAAA ATGATTATAT TTTAGACCCTTTCGTCGGAA 2300 GTGGAACTAC TGGTGTAGTA GCCAAGAGAT TGGGGCGTAA ATTTATTGGGATTGATTCTG 2360 AGAAAGAATA TCTTAAAATT GCTAAAAAAA GGCTAAATAA AGGAGCAACATATGGACTTT 2420 AATAATTACA TCGGTTTAGA ATCTGACGAT AGATTAAATG CTTTTATGGCAACACTTTCC 2480 GTAACTAATA GAACTCCCGA ATACTACGTG AACTGGGAAA AAGTTGAACGTGAAACACGA 2540 AAATTTGAAT TAGAACTAAA TACTTTAAAC TATCTCATTG GGAAAGAAGATATTTATAGT 2600 GAAGCACTTG AACTATTTAC CAATCAACCT GAATTGCTTA AAGCTATTCCTAGTTTGATT 2660 GCTAGTAGAG ATACATCTTT AGATATACTA AACATTGACG AAAATGATGATATGAGTTTT 2720 GAACAACTTA ACTTTCTTGT TATCGACGAA AATTGTATCG CTGATTATGTAGACTTTATT 2780 AACCAGGCAG GTTTACTAGA TTTTCTACAG AATAAAGCAA AACGTTCTCTGGTAGACTAT 2840 GTGTATGGTG TTGAAGCAGG GCTTGATAGC AATGCTCGAA AAAACCGAAGCGGTACAACC 2900 ATGGAGGGGA TTTTAGAACG TACTGTTTCA AAAATAGCTC AAGAGAAAGGGCTTGAATGG 2960 AAGCCACAGG CAACCGCTTC TTTTATCAAG TCTCAATGGG ACATAGAAGTCCCTGTAGAC 3020 AAATCAAAAA GACGCTTTGA TGCAGCAGTT TACTCTCGTG CGCTCAATAAGGTTTGGCTC 3080 ATAGAAACAA ATTACTACGG CGGTGGAGGA AGTAAACTCA AAGCAGTTGCTGGAGAATTT 3140 ACAGAATTGA GTCAGTTTGT AAAAACATCA AAAGATAATG TTGAATTTGTATGGGTAACA 3200 GACGGCCAAG GGTGGAAATT TTCCCGCTTA CCACTTGCAG AAGCTTTCGGACACATCGAT 3260 AACGTTTTCA ATCTAACCAT GTTGAAAGAA GGTTTCTTGT CTGATTTATTCGAAAAAGAA 3320 ATTTAAAAAG ACAGAGAATC TCTGTCTTTT TAAATTTCAA TTCCTTCCTTCTGCTAGCTA 3380 TAACTTTCCA AAAAACCTGA AAAACGGTTC TGTTGCAATT GTATGTGGGGTCGGAACTTA 3440 CTACTATATC ATGAGAAATG AAGATTAAAG TTGAAACAAA AAAACAGATTATTTTAAAAT 3500 GTAAATCTGT TTTTGTTTGG GCTGATTTTA TCACACCAAT TCTATGTTCAGAAAATGGTC 3560 ATTTTCTGGA CACTCTTCTT TTGTTATTAA AACTCTCAAA ATCATTTACATTTATTGTTC 3620 ATTAACCCAT AATTTATTCT ATGTTCATTT ATAGATATCG AATTCCTGCAGGGCCCTCCA 3680 CTAGTTCTAG AGGCG 3695 284 amino acids amino acid linearprotein not provided 2 Met Asn Leu Leu Gln Lys Asn Lys Ile Asn Leu ArgPro Phe Thr Lys 1 5 10 15 Trp Thr Gly Gly Lys Arg Gln Leu Leu Pro HisIle Gln Tyr Leu Met 20 25 30 Pro Glu Lys Tyr Asn His Phe Phe Glu Pro PheIle Gly Gly Gly Ala 35 40 45 Leu Phe Phe Glu Leu Ala Pro Gln Lys Ala ValIle Asn Asp Phe Asn 50 55 60 Ser Glu Leu Ile Asn Cys Tyr Arg Gln Met LysAsp Asn Pro Glu Gln 65 70 75 80 Leu Ile Glu Leu Leu Thr Asn His Gln ArgGlu Asn Ser Lys Glu Tyr 85 90 95 Tyr Leu Asp Leu Arg Ser Ser Asp Arg AspGly Arg Ile Asp Lys Met 100 105 110 Ser Glu Val Glu Arg Ala Ala Arg IleMet Tyr Met Leu Arg Val Asp 115 120 125 Phe Asn Gly Leu Tyr Arg Val AsnSer Lys Asn Gln Phe Asn Val Pro 130 135 140 Tyr Gly Arg Tyr Lys Asn ProLys Ile Val Asp Lys Glu Leu Ile Glu 145 150 155 160 Ser Ile Ser Glu TyrLeu Asn Asn Asn Ser Ile Lys Ile Met Ser Gly 165 170 175 Asp Phe Glu LysAla Val Lys Glu Ala Gln Asp Gly Asp Phe Val Tyr 180 185 190 Phe Asp ProPro Tyr Ile Pro Leu Ser Glu Thr Ser Ala Phe Thr Ser 195 200 205 Tyr ThrHis Glu Gly Phe Ser Tyr Glu Asp Gln Val Arg Leu Arg Asp 210 215 220 CysPhe Lys Gln Leu Asp Ser Lys Gly Val Phe Val Met Leu Ser Asn 225 230 235240 Ser Ser Ser Pro Leu Ala Glu Glu Leu Tyr Lys Asp Phe Tyr Ile His 245250 255 Lys Ile Glu Ala Thr Arg Thr Asn Gly Ala Lys Ser Ser Ser Arg Gly260 265 270 Lys Ile Thr Glu Ile Ile Val Thr Asn Tyr Gly Asn 275 280 3695base pairs nucleic acid double linear DNA (genomic) NO NO Lactococcuslactis subsp. cremoris W9 CDS 1613..2419 experimental /codon_start= 1613/product= “LlaAI -GATC- adenine methylase B” /evidence= EXPERIMENTAL/gene= “ORF” /number= 2 /standard_name= “Gene coding for M.LlaAIB”/label= m-llaAIB 3 ATATAAGATA TATAAATCAG TTCGCCTTTT TCTACTCCGTTCTAAAATCT TAAAATCAAG 60 GTCAAAAGAA AAAGTCAAAA CCATTGAATT GAGGTTCTAAAATTAAACTC CCTGCGTTGC 120 TCTTGGCTGC CGCTTGTACA CTCTGATTTT ATATTAGATACATTCTGCCA TTAAAAAGAA 180 CTCCTAACGG TCGTGGCTAC TTTGTTTAGT CTAAACGCTTTAAATAGTCC TACAAGCTCA 240 TATTTTGCCT TTTAAGCGAT TTTAAACGTG AGTTAGTAATAATTATCATG GATAAAAGAA 300 AAAGCCCTTA AATAGGCTTG TATGTAATTG ACTAAAACGTACAATTTAGC TTTTAAATAT 360 GACCCTTATT TATGACCTGC TCTAACCTCA CTATTCATCAGCATTCAAAA AAGAGGTCAA 420 AACTGTTAAG TTATGAGCTG AATAGATTTT ATTAAATTTTATTTGGTTTA AAAGACCAAT 480 TATCTATTTT TTAACAAACA CTAAAATAGA TTTTTTGGAAAACTTTGCAA CAGAACCAGC 540 AATCTGATGT TGCGAGATGG ACGTTCTTTC GGTTTTGAACCTCAAGGGGA ACACTCGTTT 600 GATAAAGCGT CTCAATGGTT GTCAGTAAAC AAACAAAAACTTTTGGAAGT GTGCTATTAT 660 AAGTCATATA AGTCGTGCGC TTTCTAATGC TTAGTGCTTTAAGATTAGGA TAGCACGACT 720 TATTTATTTT CCAATAAAAT TAACTAGCAA TTCGGGTATAATATATTTAT GAATTTATTA 780 CAAAAAAACA AGATCAACTT ACGTCCGTTT ACTAAATGGACAGGTGGGAA AAGGCAACTA 840 CTGCCACACA TTCAATACCT AATGCCAGAA AAATACAATCATTTTTTCGA ACCTTTTATT 900 GGTGGTGGCG CTTTGTTTTT TGAACTCGCT CCTCAAAAAGCAGTTATTAA CGACTTCAAT 960 TCTGAGCTTA TAAACTGTTA CCGGCAGATG AAAGATAATCCTGAGCAATT GATAGAATTG 1020 TTGACTAATC ATCAGCGGGA AAATTCTAAA GAATATTATTTAGACTTACG TTCTTCTGAT 1080 AGAGATGGAA GAATTGATAA GATGAGCGAA GTTGAACGTGCTGCTAGAAT TATGTATATG 1140 CTACGTGTTG ATTTTAATGG TTTATATCGT GTTAATTCGAAAAACCAGTT TAATGTGCCT 1200 TATGGAAGAT ATAAAAATCC TAAGATAGTT GATAAAGAATTGATTGAAAG TATTTCCGAG 1260 TACTTGAATA ACAATTCTAT TAAGATCATG AGTGGAGATTTTGAAAAAGC CGTTAAAGAA 1320 GCACAGGATG GAGATTTTGT TTATTTCGAC CCTCCATACATTCCACTTTC TGAAACTAGC 1380 GCCTTTACTT CTTATACACA CGAAGGCTTT AGCTACGAAGATCAAGTTAG GCTAAGAGAT 1440 TGTTTCAAAC AGTTAGATTC AAAAGGGGTA TTCGTCATGCTTTCAAATTC TTCAAGCCCT 1500 TTAGCGGAGG AATTATATAA AGATTTTTAC ATCCATAAAATTGAAGCTAC TCGAACAAAT 1560 GGGGCTAAAT CATCTAGTCG TGGAAAAATC ACTGAAATCATCGTAACCAA TT ATG 1615 Met 285 GCA ATT AAC GAA TAT AAG TAT GGA GGT GTTTTA ATG ATA AAA CCA TAC 1663 Ala Ile Asn Glu Tyr Lys Tyr Gly Gly Val LeuMet Ile Lys Pro Tyr 290 295 300 TAT GAA AAA GAA AAC GCA ATT CTC GTT CACGCA GAT TCA TTT AAA TTA 1711 Tyr Glu Lys Glu Asn Ala Ile Leu Val His AlaAsp Ser Phe Lys Leu 305 310 315 TTA GAA AAA ATT AAA CCT GAA AGC ATG GACATG ATA TTT GCT GAC CCT 1759 Leu Glu Lys Ile Lys Pro Glu Ser Met Asp MetIle Phe Ala Asp Pro 320 325 330 CCT TAC TTT TTA AGT AAT GGA GGA ATG TCAAAT TCA GGT GGT CAA ATT 1807 Pro Tyr Phe Leu Ser Asn Gly Gly Met Ser AsnSer Gly Gly Gln Ile 335 340 345 GTT TCT GTT GAT AAA GGG GAT TGG GAT AAAATT TCT TCA TTT GAA GAA 1855 Val Ser Val Asp Lys Gly Asp Trp Asp Lys IleSer Ser Phe Glu Glu 350 355 360 365 AAA CAT GAC TTT AAT AGA CGT TGG ATTAGG TTA GCA AGA TTG GTT TTA 1903 Lys His Asp Phe Asn Arg Arg Trp Ile ArgLeu Ala Arg Leu Val Leu 370 375 380 AAA CCC AAC GGA ACT ATT TGG GTT TCCGGA AGC CTT CAT AAC ATA TAT 1951 Lys Pro Asn Gly Thr Ile Trp Val Ser GlySer Leu His Asn Ile Tyr 385 390 395 TCT GTC GGG ATG GCG CTG GAA CAG GAAGGT TTC AAA ATC TTA AAT AAT 1999 Ser Val Gly Met Ala Leu Glu Gln Glu GlyPhe Lys Ile Leu Asn Asn 400 405 410 ATA ACT TGG CAA AAG ACA AAT CCT GCACCT AAT CTA TCA TGT CGG TAC 2047 Ile Thr Trp Gln Lys Thr Asn Pro Ala ProAsn Leu Ser Cys Arg Tyr 415 420 425 TTC ACC CAC TCT ACA GAG ACA ATT TTATGG GCA AGA AAG AAC GAT AAA 2095 Phe Thr His Ser Thr Glu Thr Ile Leu TrpAla Arg Lys Asn Asp Lys 430 435 440 445 AAA TCT CGC CAT TAT TAT AAC TATGAA TTG ATG AAA GAG TTT AAT GAC 2143 Lys Ser Arg His Tyr Tyr Asn Tyr GluLeu Met Lys Glu Phe Asn Asp 450 455 460 GGG AAA CAA ATG AAA GAT GTT TGGACA GGT AGT CTG ACA AAA AAA TCA 2191 Gly Lys Gln Met Lys Asp Val Trp ThrGly Ser Leu Thr Lys Lys Ser 465 470 475 GAA AAA TGG GCT GGG AAA CAT CCAACT CAG AAG CCA GAG TAT ATT TTA 2239 Glu Lys Trp Ala Gly Lys His Pro ThrGln Lys Pro Glu Tyr Ile Leu 480 485 490 GAA CGG ATA ATC TTA GCT AGT ACAAAG GAA AAT GAT TAT ATT TTA GAC 2287 Glu Arg Ile Ile Leu Ala Ser Thr LysGlu Asn Asp Tyr Ile Leu Asp 495 500 505 CCT TTC GTC GGA AGT GGA ACT ACTGGT GTA GTA GCC AAG AGA TTG GGG 2335 Pro Phe Val Gly Ser Gly Thr Thr GlyVal Val Ala Lys Arg Leu Gly 510 515 520 525 CGT AAA TTT ATT GGG ATT GATTCT GAG AAA GAA TAT CTT AAA ATT GCT 2383 Arg Lys Phe Ile Gly Ile Asp SerGlu Lys Glu Tyr Leu Lys Ile Ala 530 535 540 AAA AAA AGG CTA AAT AAA GGAGCA ACA TAT GGA CTT TAATAATTAC 2429 Lys Lys Arg Leu Asn Lys Gly Ala ThrTyr Gly Leu 545 550 ATCGGTTTAG AATCTGACGA TAGATTAAAT GCTTTTATGGCAACACTTTC CGTAACTAAT 2489 AGAACTCCCG AATACTACGT GAACTGGGAA AAAGTTGAACGTGAAACACG AAAATTTGAA 2549 TTAGAACTAA ATACTTTAAA CTATCTCATT GGGAAAGAAGATATTTATAG TGAAGCACTT 2609 GAACTATTTA CCAATCAACC TGAATTGCTT AAAGCTATTCCTAGTTTGAT TGCTAGTAGA 2669 GATACATCTT TAGATATACT AAACATTGAC GAAAATGATGATATGAGTTT TGAACAACTT 2729 AACTTTCTTG TTATCGACGA AAATTGTATC GCTGATTATGTAGACTTTAT TAACCAGGCA 2789 GGTTTACTAG ATTTTCTACA GAATAAAGCA AAACGTTCTCTGGTAGACTA TGTGTATGGT 2849 GTTGAAGCAG GGCTTGATAG CAATGCTCGA AAAAACCGAAGCGGTACAAC CATGGAGGGG 2909 ATTTTAGAAC GTACTGTTTC AAAAATAGCT CAAGAGAAAGGGCTTGAATG GAAGCCACAG 2969 GCAACCGCTT CTTTTATCAA GTCTCAATGG GACATAGAAGTCCCTGTAGA CAAATCAAAA 3029 AGACGCTTTG ATGCAGCAGT TTACTCTCGT GCGCTCAATAAGGTTTGGCT CATAGAAACA 3089 AATTACTACG GCGGTGGAGG AAGTAAACTC AAAGCAGTTGCTGGAGAATT TACAGAATTG 3149 AGTCAGTTTG TAAAAACATC AAAAGATAAT GTTGAATTTGTATGGGTAAC AGACGGCCAA 3209 GGGTGGAAAT TTTCCCGCTT ACCACTTGCA GAAGCTTTCGGACACATCGA TAACGTTTTC 3269 AATCTAACCA TGTTGAAAGA AGGTTTCTTG TCTGATTTATTCGAAAAAGA AATTTAAAAA 3329 GACAGAGAAT CTCTGTCTTT TTAAATTTCA ATTCCTTCCTTCTGCTAGCT ATAACTTTCC 3389 AAAAAACCTG AAAAACGGTT CTGTTGCAAT TGTATGTGGGGTCGGAACTT ACTACTATAT 3449 CATGAGAAAT GAAGATTAAA GTTGAAACAA AAAAACAGATTATTTTAAAA TGTAAATCTG 3509 TTTTTGTTTG GGCTGATTTT ATCACACCAA TTCTATGTTCAGAAAATGGT CATTTTCTGG 3569 ACACTCTTCT TTTGTTATTA AAACTCTCAA AATCATTTACATTTATTGTT CATTAACCCA 3629 TAATTTATTC TATGTTCATT TATAGATATC GAATTCCTGCAGGGCCCTCC ACTAGTTCTA 3689 GAGGCG 3695 269 amino acids amino acid linearprotein not provided 4 Met Ala Ile Asn Glu Tyr Lys Tyr Gly Gly Val LeuMet Ile Lys Pro 1 5 10 15 Tyr Tyr Glu Lys Glu Asn Ala Ile Leu Val HisAla Asp Ser Phe Lys 20 25 30 Leu Leu Glu Lys Ile Lys Pro Glu Ser Met AspMet Ile Phe Ala Asp 35 40 45 Pro Pro Tyr Phe Leu Ser Asn Gly Gly Met SerAsn Ser Gly Gly Gln 50 55 60 Ile Val Ser Val Asp Lys Gly Asp Trp Asp LysIle Ser Ser Phe Glu 65 70 75 80 Glu Lys His Asp Phe Asn Arg Arg Trp IleArg Leu Ala Arg Leu Val 85 90 95 Leu Lys Pro Asn Gly Thr Ile Trp Val SerGly Ser Leu His Asn Ile 100 105 110 Tyr Ser Val Gly Met Ala Leu Glu GlnGlu Gly Phe Lys Ile Leu Asn 115 120 125 Asn Ile Thr Trp Gln Lys Thr AsnPro Ala Pro Asn Leu Ser Cys Arg 130 135 140 Tyr Phe Thr His Ser Thr GluThr Ile Leu Trp Ala Arg Lys Asn Asp 145 150 155 160 Lys Lys Ser Arg HisTyr Tyr Asn Tyr Glu Leu Met Lys Glu Phe Asn 165 170 175 Asp Gly Lys GlnMet Lys Asp Val Trp Thr Gly Ser Leu Thr Lys Lys 180 185 190 Ser Glu LysTrp Ala Gly Lys His Pro Thr Gln Lys Pro Glu Tyr Ile 195 200 205 Leu GluArg Ile Ile Leu Ala Ser Thr Lys Glu Asn Asp Tyr Ile Leu 210 215 220 AspPro Phe Val Gly Ser Gly Thr Thr Gly Val Val Ala Lys Arg Leu 225 230 235240 Gly Arg Lys Phe Ile Gly Ile Asp Ser Glu Lys Glu Tyr Leu Lys Ile 245250 255 Ala Lys Lys Arg Leu Asn Lys Gly Ala Thr Tyr Gly Leu 260 265 3695base pairs nucleic acid double linear DNA (genomic) NO NO Lactococcuslactis subsp. cremoris W9 CDS 1649..2419 experimental /codon_start= 1649/product= “LlaAI -GATC-adenine methylase B” /evidence= EXPERIMENTAL/gene= “ORF” /number= 2 /standard_name= “Gene coding for M.LlaAIB”/label= m-llaAIB 5 ATATAAGATA TATAAATCAG TTCGCCTTTT TCTACTCCGTTCTAAAATCT TAAAATCAAG 60 GTCAAAAGAA AAAGTCAAAA CCATTGAATT GAGGTTCTAAAATTAAACTC CCTGCGTTGC 120 TCTTGGCTGC CGCTTGTACA CTCTGATTTT ATATTAGATACATTCTGCCA TTAAAAAGAA 180 CTCCTAACGG TCGTGGCTAC TTTGTTTAGT CTAAACGCTTTAAATAGTCC TACAAGCTCA 240 TATTTTGCCT TTTAAGCGAT TTTAAACGTG AGTTAGTAATAATTATCATG GATAAAAGAA 300 AAAGCCCTTA AATAGGCTTG TATGTAATTG ACTAAAACGTACAATTTAGC TTTTAAATAT 360 GACCCTTATT TATGACCTGC TCTAACCTCA CTATTCATCAGCATTCAAAA AAGAGGTCAA 420 AACTGTTAAG TTATGAGCTG AATAGATTTT ATTAAATTTTATTTGGTTTA AAAGACCAAT 480 TATCTATTTT TTAACAAACA CTAAAATAGA TTTTTTGGAAAACTTTGCAA CAGAACCAGC 540 AATCTGATGT TGCGAGATGG ACGTTCTTTC GGTTTTGAACCTCAAGGGGA ACACTCGTTT 600 GATAAAGCGT CTCAATGGTT GTCAGTAAAC AAACAAAAACTTTTGGAAGT GTGCTATTAT 660 AAGTCATATA AGTCGTGCGC TTTCTAATGC TTAGTGCTTTAAGATTAGGA TAGCACGACT 720 TATTTATTTT CCAATAAAAT TAACTAGCAA TTCGGGTATAATATATTTAT GAATTTATTA 780 CAAAAAAACA AGATCAACTT ACGTCCGTTT ACTAAATGGACAGGTGGGAA AAGGCAACTA 840 CTGCCACACA TTCAATACCT AATGCCAGAA AAATACAATCATTTTTTCGA ACCTTTTATT 900 GGTGGTGGCG CTTTGTTTTT TGAACTCGCT CCTCAAAAAGCAGTTATTAA CGACTTCAAT 960 TCTGAGCTTA TAAACTGTTA CCGGCAGATG AAAGATAATCCTGAGCAATT GATAGAATTG 1020 TTGACTAATC ATCAGCGGGA AAATTCTAAA GAATATTATTTAGACTTACG TTCTTCTGAT 1080 AGAGATGGAA GAATTGATAA GATGAGCGAA GTTGAACGTGCTGCTAGAAT TATGTATATG 1140 CTACGTGTTG ATTTTAATGG TTTATATCGT GTTAATTCGAAAAACCAGTT TAATGTGCCT 1200 TATGGAAGAT ATAAAAATCC TAAGATAGTT GATAAAGAATTGATTGAAAG TATTTCCGAG 1260 TACTTGAATA ACAATTCTAT TAAGATCATG AGTGGAGATTTTGAAAAAGC CGTTAAAGAA 1320 GCACAGGATG GAGATTTTGT TTATTTCGAC CCTCCATACATTCCACTTTC TGAAACTAGC 1380 GCCTTTACTT CTTATACACA CGAAGGCTTT AGCTACGAAGATCAAGTTAG GCTAAGAGAT 1440 TGTTTCAAAC AGTTAGATTC AAAAGGGGTA TTCGTCATGCTTTCAAATTC TTCAAGCCCT 1500 TTAGCGGAGG AATTATATAA AGATTTTTAC ATCCATAAAATTGAAGCTAC TCGAACAAAT 1560 GGGGCTAAAT CATCTAGTCG TGGAAAAATC ACTGAAATCATCGTAACCAA TTATGGCAAT 1620 TAACGAATAT AAGTATGGAG GTGTTTTA ATG ATA AAACCA TAC TAT GAA AAA 1672 Met Ile Lys Pro Tyr Tyr Glu Lys 270 275 GAA AACGCA ATT CTC GTT CAC GCA GAT TCA TTT AAA TTA TTA GAA AAA 1720 Glu Asn AlaIle Leu Val His Ala Asp Ser Phe Lys Leu Leu Glu Lys 280 285 290 ATT AAACCT GAA AGC ATG GAC ATG ATA TTT GCT GAC CCT CCT TAC TTT 1768 Ile Lys ProGlu Ser Met Asp Met Ile Phe Ala Asp Pro Pro Tyr Phe 295 300 305 TTA AGTAAT GGA GGA ATG TCA AAT TCA GGT GGT CAA ATT GTT TCT GTT 1816 Leu Ser AsnGly Gly Met Ser Asn Ser Gly Gly Gln Ile Val Ser Val 310 315 320 325 GATAAA GGG GAT TGG GAT AAA ATT TCT TCA TTT GAA GAA AAA CAT GAC 1864 Asp LysGly Asp Trp Asp Lys Ile Ser Ser Phe Glu Glu Lys His Asp 330 335 340 TTTAAT AGA CGT TGG ATT AGG TTA GCA AGA TTG GTT TTA AAA CCC AAC 1912 Phe AsnArg Arg Trp Ile Arg Leu Ala Arg Leu Val Leu Lys Pro Asn 345 350 355 GGAACT ATT TGG GTT TCC GGA AGC CTT CAT AAC ATA TAT TCT GTC GGG 1960 Gly ThrIle Trp Val Ser Gly Ser Leu His Asn Ile Tyr Ser Val Gly 360 365 370 ATGGCG CTG GAA CAG GAA GGT TTC AAA ATC TTA AAT AAT ATA ACT TGG 2008 Met AlaLeu Glu Gln Glu Gly Phe Lys Ile Leu Asn Asn Ile Thr Trp 375 380 385 CAAAAG ACA AAT CCT GCA CCT AAT CTA TCA TGT CGG TAC TTC ACC CAC 2056 Gln LysThr Asn Pro Ala Pro Asn Leu Ser Cys Arg Tyr Phe Thr His 390 395 400 405TCT ACA GAG ACA ATT TTA TGG GCA AGA AAG AAC GAT AAA AAA TCT CGC 2104 SerThr Glu Thr Ile Leu Trp Ala Arg Lys Asn Asp Lys Lys Ser Arg 410 415 420CAT TAT TAT AAC TAT GAA TTG ATG AAA GAG TTT AAT GAC GGG AAA CAA 2152 HisTyr Tyr Asn Tyr Glu Leu Met Lys Glu Phe Asn Asp Gly Lys Gln 425 430 435ATG AAA GAT GTT TGG ACA GGT AGT CTG ACA AAA AAA TCA GAA AAA TGG 2200 MetLys Asp Val Trp Thr Gly Ser Leu Thr Lys Lys Ser Glu Lys Trp 440 445 450GCT GGG AAA CAT CCA ACT CAG AAG CCA GAG TAT ATT TTA GAA CGG ATA 2248 AlaGly Lys His Pro Thr Gln Lys Pro Glu Tyr Ile Leu Glu Arg Ile 455 460 465ATC TTA GCT AGT ACA AAG GAA AAT GAT TAT ATT TTA GAC CCT TTC GTC 2296 IleLeu Ala Ser Thr Lys Glu Asn Asp Tyr Ile Leu Asp Pro Phe Val 470 475 480485 GGA AGT GGA ACT ACT GGT GTA GTA GCC AAG AGA TTG GGG CGT AAA TTT 2344Gly Ser Gly Thr Thr Gly Val Val Ala Lys Arg Leu Gly Arg Lys Phe 490 495500 ATT GGG ATT GAT TCT GAG AAA GAA TAT CTT AAA ATT GCT AAA AAA AGG 2392Ile Gly Ile Asp Ser Glu Lys Glu Tyr Leu Lys Ile Ala Lys Lys Arg 505 510515 CTA AAT AAA GGA GCA ACA TAT GGA CTT TAATAATTAC ATCGGTTTAG 2439 LeuAsn Lys Gly Ala Thr Tyr Gly Leu 520 525 AATCTGACGA TAGATTAAAT GCTTTTATGGCAACACTTTC CGTAACTAAT AGAACTCCCG 2499 AATACTACGT GAACTGGGAA AAAGTTGAACGTGAAACACG AAAATTTGAA TTAGAACTAA 2559 ATACTTTAAA CTATCTCATT GGGAAAGAAGATATTTATAG TGAAGCACTT GAACTATTTA 2619 CCAATCAACC TGAATTGCTT AAAGCTATTCCTAGTTTGAT TGCTAGTAGA GATACATCTT 2679 TAGATATACT AAACATTGAC GAAAATGATGATATGAGTTT TGAACAACTT AACTTTCTTG 2739 TTATCGACGA AAATTGTATC GCTGATTATGTAGACTTTAT TAACCAGGCA GGTTTACTAG 2799 ATTTTCTACA GAATAAAGCA AAACGTTCTCTGGTAGACTA TGTGTATGGT GTTGAAGCAG 2859 GGCTTGATAG CAATGCTCGA AAAAACCGAAGCGGTACAAC CATGGAGGGG ATTTTAGAAC 2919 GTACTGTTTC AAAAATAGCT CAAGAGAAAGGGCTTGAATG GAAGCCACAG GCAACCGCTT 2979 CTTTTATCAA GTCTCAATGG GACATAGAAGTCCCTGTAGA CAAATCAAAA AGACGCTTTG 3039 ATGCAGCAGT TTACTCTCGT GCGCTCAATAAGGTTTGGCT CATAGAAACA AATTACTACG 3099 GCGGTGGAGG AAGTAAACTC AAAGCAGTTGCTGGAGAATT TACAGAATTG AGTCAGTTTG 3159 TAAAAACATC AAAAGATAAT GTTGAATTTGTATGGGTAAC AGACGGCCAA GGGTGGAAAT 3219 TTTCCCGCTT ACCACTTGCA GAAGCTTTCGGACACATCGA TAACGTTTTC AATCTAACCA 3279 TGTTGAAAGA AGGTTTCTTG TCTGATTTATTCGAAAAAGA AATTTAAAAA GACAGAGAAT 3339 CTCTGTCTTT TTAAATTTCA ATTCCTTCCTTCTGCTAGCT ATAACTTTCC AAAAAACCTG 3399 AAAAACGGTT CTGTTGCAAT TGTATGTGGGGTCGGAACTT ACTACTATAT CATGAGAAAT 3459 GAAGATTAAA GTTGAAACAA AAAAACAGATTATTTTAAAA TGTAAATCTG TTTTTGTTTG 3519 GGCTGATTTT ATCACACCAA TTCTATGTTCAGAAAATGGT CATTTTCTGG ACACTCTTCT 3579 TTTGTTATTA AAACTCTCAA AATCATTTACATTTATTGTT CATTAACCCA TAATTTATTC 3639 TATGTTCATT TATAGATATC GAATTCCTGCAGGGCCCTCC ACTAGTTCTA GAGGCG 3695 257 amino acids amino acid linearprotein not provided 6 Met Ile Lys Pro Tyr Tyr Glu Lys Glu Asn Ala IleLeu Val His Ala 1 5 10 15 Asp Ser Phe Lys Leu Leu Glu Lys Ile Lys ProGlu Ser Met Asp Met 20 25 30 Ile Phe Ala Asp Pro Pro Tyr Phe Leu Ser AsnGly Gly Met Ser Asn 35 40 45 Ser Gly Gly Gln Ile Val Ser Val Asp Lys GlyAsp Trp Asp Lys Ile 50 55 60 Ser Ser Phe Glu Glu Lys His Asp Phe Asn ArgArg Trp Ile Arg Leu 65 70 75 80 Ala Arg Leu Val Leu Lys Pro Asn Gly ThrIle Trp Val Ser Gly Ser 85 90 95 Leu His Asn Ile Tyr Ser Val Gly Met AlaLeu Glu Gln Glu Gly Phe 100 105 110 Lys Ile Leu Asn Asn Ile Thr Trp GlnLys Thr Asn Pro Ala Pro Asn 115 120 125 Leu Ser Cys Arg Tyr Phe Thr HisSer Thr Glu Thr Ile Leu Trp Ala 130 135 140 Arg Lys Asn Asp Lys Lys SerArg His Tyr Tyr Asn Tyr Glu Leu Met 145 150 155 160 Lys Glu Phe Asn AspGly Lys Gln Met Lys Asp Val Trp Thr Gly Ser 165 170 175 Leu Thr Lys LysSer Glu Lys Trp Ala Gly Lys His Pro Thr Gln Lys 180 185 190 Pro Glu TyrIle Leu Glu Arg Ile Ile Leu Ala Ser Thr Lys Glu Asn 195 200 205 Asp TyrIle Leu Asp Pro Phe Val Gly Ser Gly Thr Thr Gly Val Val 210 215 220 AlaLys Arg Leu Gly Arg Lys Phe Ile Gly Ile Asp Ser Glu Lys Glu 225 230 235240 Tyr Leu Lys Ile Ala Lys Lys Arg Leu Asn Lys Gly Ala Thr Tyr Gly 245250 255 Leu 3695 base pairs nucleic acid double linear DNA (genomic) NONO Lactococcus lactis subsp. cremoris W9 CDS 2412..3323 experimental/codon_start= 2412 /product= “LlaAI restriction endonuclease” /evidence=EXPERIMENTAL /gene= “ORF” /number= 3 /standard_name= “Gene coding forLlaAI restriction endonuclease” /label= r-llaAI 7 ATATAAGATA TATAAATCAGTTCGCCTTTT TCTACTCCGT TCTAAAATCT TAAAATCAAG 60 GTCAAAAGAA AAAGTCAAAACCATTGAATT GAGGTTCTAA AATTAAACTC CCTGCGTTGC 120 TCTTGGCTGC CGCTTGTACACTCTGATTTT ATATTAGATA CATTCTGCCA TTAAAAAGAA 180 CTCCTAACGG TCGTGGCTACTTTGTTTAGT CTAAACGCTT TAAATAGTCC TACAAGCTCA 240 TATTTTGCCT TTTAAGCGATTTTAAACGTG AGTTAGTAAT AATTATCATG GATAAAAGAA 300 AAAGCCCTTA AATAGGCTTGTATGTAATTG ACTAAAACGT ACAATTTAGC TTTTAAATAT 360 GACCCTTATT TATGACCTGCTCTAACCTCA CTATTCATCA GCATTCAAAA AAGAGGTCAA 420 AACTGTTAAG TTATGAGCTGAATAGATTTT ATTAAATTTT ATTTGGTTTA AAAGACCAAT 480 TATCTATTTT TTAACAAACACTAAAATAGA TTTTTTGGAA AACTTTGCAA CAGAACCAGC 540 AATCTGATGT TGCGAGATGGACGTTCTTTC GGTTTTGAAC CTCAAGGGGA ACACTCGTTT 600 GATAAAGCGT CTCAATGGTTGTCAGTAAAC AAACAAAAAC TTTTGGAAGT GTGCTATTAT 660 AAGTCATATA AGTCGTGCGCTTTCTAATGC TTAGTGCTTT AAGATTAGGA TAGCACGACT 720 TATTTATTTT CCAATAAAATTAACTAGCAA TTCGGGTATA ATATATTTAT GAATTTATTA 780 CAAAAAAACA AGATCAACTTACGTCCGTTT ACTAAATGGA CAGGTGGGAA AAGGCAACTA 840 CTGCCACACA TTCAATACCTAATGCCAGAA AAATACAATC ATTTTTTCGA ACCTTTTATT 900 GGTGGTGGCG CTTTGTTTTTTGAACTCGCT CCTCAAAAAG CAGTTATTAA CGACTTCAAT 960 TCTGAGCTTA TAAACTGTTACCGGCAGATG AAAGATAATC CTGAGCAATT GATAGAATTG 1020 TTGACTAATC ATCAGCGGGAAAATTCTAAA GAATATTATT TAGACTTACG TTCTTCTGAT 1080 AGAGATGGAA GAATTGATAAGATGAGCGAA GTTGAACGTG CTGCTAGAAT TATGTATATG 1140 CTACGTGTTG ATTTTAATGGTTTATATCGT GTTAATTCGA AAAACCAGTT TAATGTGCCT 1200 TATGGAAGAT ATAAAAATCCTAAGATAGTT GATAAAGAAT TGATTGAAAG TATTTCCGAG 1260 TACTTGAATA ACAATTCTATTAAGATCATG AGTGGAGATT TTGAAAAAGC CGTTAAAGAA 1320 GCACAGGATG GAGATTTTGTTTATTTCGAC CCTCCATACA TTCCACTTTC TGAAACTAGC 1380 GCCTTTACTT CTTATACACACGAAGGCTTT AGCTACGAAG ATCAAGTTAG GCTAAGAGAT 1440 TGTTTCAAAC AGTTAGATTCAAAAGGGGTA TTCGTCATGC TTTCAAATTC TTCAAGCCCT 1500 TTAGCGGAGG AATTATATAAAGATTTTTAC ATCCATAAAA TTGAAGCTAC TCGAACAAAT 1560 GGGGCTAAAT CATCTAGTCGTGGAAAAATC ACTGAAATCA TCGTAACCAA TTATGGCAAT 1620 TAACGAATAT AAGTATGGAGGTGTTTTAAT GATAAAACCA TACTATGAAA AAGAAAACGC 1680 AATTCTCGTT CACGCAGATTCATTTAAATT ATTAGAAAAA ATTAAACCTG AAAGCATGGA 1740 CATGATATTT GCTGACCCTCCTTACTTTTT AAGTAATGGA GGAATGTCAA ATTCAGGTGG 1800 TCAAATTGTT TCTGTTGATAAAGGGGATTG GGATAAAATT TCTTCATTTG AAGAAAAACA 1860 TGACTTTAAT AGACGTTGGATTAGGTTAGC AAGATTGGTT TTAAAACCCA ACGGAACTAT 1920 TTGGGTTTCC GGAAGCCTTCATAACATATA TTCTGTCGGG ATGGCGCTGG AACAGGAAGG 1980 TTTCAAAATC TTAAATAATATAACTTGGCA AAAGACAAAT CCTGCACCTA ATCTATCATG 2040 TCGGTACTTC ACCCACTCTACAGAGACAAT TTTATGGGCA AGAAAGAACG ATAAAAAATC 2100 TCGCCATTAT TATAACTATGAATTGATGAA AGAGTTTAAT GACGGGAAAC AAATGAAAGA 2160 TGTTTGGACA GGTAGTCTGACAAAAAAATC AGAAAAATGG GCTGGGAAAC ATCCAACTCA 2220 GAAGCCAGAG TATATTTTAGAACGGATAAT CTTAGCTAGT ACAAAGGAAA ATGATTATAT 2280 TTTAGACCCT TTCGTCGGAAGTGGAACTAC TGGTGTAGTA GCCAAGAGAT TGGGGCGTAA 2340 ATTTATTGGG ATTGATTCTGAGAAAGAATA TCTTAAAATT GCTAAAAAAA GGCTAAATAA 2400 AGGAGCAACA T ATG GACTTT AAT AAT TAC ATC GGT TTA GAA TCT GAC GAT 2450 Met Asp Phe Asn Asn TyrIle Gly Leu Glu Ser Asp Asp 260 265 270 AGA TTA AAT GCT TTT ATG GCA ACACTT TCC GTA ACT AAT AGA ACT CCC 2498 Arg Leu Asn Ala Phe Met Ala Thr LeuSer Val Thr Asn Arg Thr Pro 275 280 285 GAA TAC TAC GTG AAC TGG GAA AAAGTT GAA CGT GAA ACA CGA AAA TTT 2546 Glu Tyr Tyr Val Asn Trp Glu Lys ValGlu Arg Glu Thr Arg Lys Phe 290 295 300 GAA TTA GAA CTA AAT ACT TTA AACTAT CTC ATT GGG AAA GAA GAT ATT 2594 Glu Leu Glu Leu Asn Thr Leu Asn TyrLeu Ile Gly Lys Glu Asp Ile 305 310 315 TAT AGT GAA GCA CTT GAA CTA TTTACC AAT CAA CCT GAA TTG CTT AAA 2642 Tyr Ser Glu Ala Leu Glu Leu Phe ThrAsn Gln Pro Glu Leu Leu Lys 320 325 330 GCT ATT CCT AGT TTG ATT GCT AGTAGA GAT ACA TCT TTA GAT ATA CTA 2690 Ala Ile Pro Ser Leu Ile Ala Ser ArgAsp Thr Ser Leu Asp Ile Leu 335 340 345 350 AAC ATT GAC GAA AAT GAT GATATG AGT TTT GAA CAA CTT AAC TTT CTT 2738 Asn Ile Asp Glu Asn Asp Asp MetSer Phe Glu Gln Leu Asn Phe Leu 355 360 365 GTT ATC GAC GAA AAT TGT ATCGCT GAT TAT GTA GAC TTT ATT AAC CAG 2786 Val Ile Asp Glu Asn Cys Ile AlaAsp Tyr Val Asp Phe Ile Asn Gln 370 375 380 GCA GGT TTA CTA GAT TTT CTACAG AAT AAA GCA AAA CGT TCT CTG GTA 2834 Ala Gly Leu Leu Asp Phe Leu GlnAsn Lys Ala Lys Arg Ser Leu Val 385 390 395 GAC TAT GTG TAT GGT GTT GAAGCA GGG CTT GAT AGC AAT GCT CGA AAA 2882 Asp Tyr Val Tyr Gly Val Glu AlaGly Leu Asp Ser Asn Ala Arg Lys 400 405 410 AAC CGA AGC GGT ACA ACC ATGGAG GGG ATT TTA GAA CGT ACT GTT TCA 2930 Asn Arg Ser Gly Thr Thr Met GluGly Ile Leu Glu Arg Thr Val Ser 415 420 425 430 AAA ATA GCT CAA GAG AAAGGG CTT GAA TGG AAG CCA CAG GCA ACC GCT 2978 Lys Ile Ala Gln Glu Lys GlyLeu Glu Trp Lys Pro Gln Ala Thr Ala 435 440 445 TCT TTT ATC AAG TCT CAATGG GAC ATA GAA GTC CCT GTA GAC AAA TCA 3026 Ser Phe Ile Lys Ser Gln TrpAsp Ile Glu Val Pro Val Asp Lys Ser 450 455 460 AAA AGA CGC TTT GAT GCAGCA GTT TAC TCT CGT GCG CTC AAT AAG GTT 3074 Lys Arg Arg Phe Asp Ala AlaVal Tyr Ser Arg Ala Leu Asn Lys Val 465 470 475 TGG CTC ATA GAA ACA AATTAC TAC GGC GGT GGA GGA AGT AAA CTC AAA 3122 Trp Leu Ile Glu Thr Asn TyrTyr Gly Gly Gly Gly Ser Lys Leu Lys 480 485 490 GCA GTT GCT GGA GAA TTTACA GAA TTG AGT CAG TTT GTA AAA ACA TCA 3170 Ala Val Ala Gly Glu Phe ThrGlu Leu Ser Gln Phe Val Lys Thr Ser 495 500 505 510 AAA GAT AAT GTT GAATTT GTA TGG GTA ACA GAC GGC CAA GGG TGG AAA 3218 Lys Asp Asn Val Glu PheVal Trp Val Thr Asp Gly Gln Gly Trp Lys 515 520 525 TTT TCC CGC TTA CCACTT GCA GAA GCT TTC GGA CAC ATC GAT AAC GTT 3266 Phe Ser Arg Leu Pro LeuAla Glu Ala Phe Gly His Ile Asp Asn Val 530 535 540 TTC AAT CTA ACC ATGTTG AAA GAA GGT TTC TTG TCT GAT TTA TTC GAA 3314 Phe Asn Leu Thr Met LeuLys Glu Gly Phe Leu Ser Asp Leu Phe Glu 545 550 555 AAA GAA ATTTAAAAAGACA GAGAATCTCT GTCTTTTTAA ATTTCAATTC 3363 Lys Glu Ile 560CTTCCTTCTG CTAGCTATAA CTTTCCAAAA AACCTGAAAA ACGGTTCTGT TGCAATTGTA 3423TGTGGGGTCG GAACTTACTA CTATATCATG AGAAATGAAG ATTAAAGTTG AAACAAAAAA 3483ACAGATTATT TTAAAATGTA AATCTGTTTT TGTTTGGGCT GATTTTATCA CACCAATTCT 3543ATGTTCAGAA AATGGTCATT TTCTGGACAC TCTTCTTTTG TTATTAAAAC TCTCAAAATC 3603ATTTACATTT ATTGTTCATT AACCCATAAT TTATTCTATG TTCATTTATA GATATCGAAT 3663TCCTGCAGGG CCCTCCACTA GTTCTAGAGG CG 3695 304 amino acids amino acidlinear protein not provided 8 Met Asp Phe Asn Asn Tyr Ile Gly Leu GluSer Asp Asp Arg Leu Asn 1 5 10 15 Ala Phe Met Ala Thr Leu Ser Val ThrAsn Arg Thr Pro Glu Tyr Tyr 20 25 30 Val Asn Trp Glu Lys Val Glu Arg GluThr Arg Lys Phe Glu Leu Glu 35 40 45 Leu Asn Thr Leu Asn Tyr Leu Ile GlyLys Glu Asp Ile Tyr Ser Glu 50 55 60 Ala Leu Glu Leu Phe Thr Asn Gln ProGlu Leu Leu Lys Ala Ile Pro 65 70 75 80 Ser Leu Ile Ala Ser Arg Asp ThrSer Leu Asp Ile Leu Asn Ile Asp 85 90 95 Glu Asn Asp Asp Met Ser Phe GluGln Leu Asn Phe Leu Val Ile Asp 100 105 110 Glu Asn Cys Ile Ala Asp TyrVal Asp Phe Ile Asn Gln Ala Gly Leu 115 120 125 Leu Asp Phe Leu Gln AsnLys Ala Lys Arg Ser Leu Val Asp Tyr Val 130 135 140 Tyr Gly Val Glu AlaGly Leu Asp Ser Asn Ala Arg Lys Asn Arg Ser 145 150 155 160 Gly Thr ThrMet Glu Gly Ile Leu Glu Arg Thr Val Ser Lys Ile Ala 165 170 175 Gln GluLys Gly Leu Glu Trp Lys Pro Gln Ala Thr Ala Ser Phe Ile 180 185 190 LysSer Gln Trp Asp Ile Glu Val Pro Val Asp Lys Ser Lys Arg Arg 195 200 205Phe Asp Ala Ala Val Tyr Ser Arg Ala Leu Asn Lys Val Trp Leu Ile 210 215220 Glu Thr Asn Tyr Tyr Gly Gly Gly Gly Ser Lys Leu Lys Ala Val Ala 225230 235 240 Gly Glu Phe Thr Glu Leu Ser Gln Phe Val Lys Thr Ser Lys AspAsn 245 250 255 Val Glu Phe Val Trp Val Thr Asp Gly Gln Gly Trp Lys PheSer Arg 260 265 270 Leu Pro Leu Ala Glu Ala Phe Gly His Ile Asp Asn ValPhe Asn Leu 275 280 285 Thr Met Leu Lys Glu Gly Phe Leu Ser Asp Leu PheGlu Lys Glu Ile 290 295 300 3706 base pairs nucleic acid double linearDNA (genomic) NO NO Lactococcus lactis subsp. cremoris W56 CDScomplement (422..2161) experimental /codon_start= 422 /product= “LlaBImethylase” /evidence= EXPERIMENTAL /gene= “ORF” /number= 1/standard_name= “Gene coding for LlaBI methylase” /label= m-llaBI CDS2464..3360 experimental /codon_start= 2464 /product= “LlaBIendonuclease” /evidence= EXPERIMENTAL /gene= “ORF” /number= 2/standard_name= “Gene coding for LlaBI endonuclease” /label= r-llaBI 9GAATTCGCAA GGTCTTTTAT AGATATAATT CCTAGCTTAT TTAAACGTTT CTCAGTTCGT 60TTTCCAATGC CCCAGAAGGC AGTAATCTTG GACTAATGTC AAATAAATAG ACACGAAAAA 120GTAAATAATA TACCCGAATT GCTAGTTAAT TTCATTGGAA AATAAATAAG TCGTGCTATC 180CTAATCTTAA ACCACTAAGC ATTAGAAAGC GCACGACTTA TAACACTGAA AAGCTTTCGG 240TTTCTAGTAT TATGCTCGGT CTTGGAGGTT TGGCAAGCTC TATGTTTGCT ACGCTTCGTG 300GCGCAAACGA CCTTGTTGGG GGAGTGTTTC ACTTCCCCCG AAACCCCCTT AAAAAACTGT 360CAAAACGTAG CCGTTTTGTA TTAAAAAAGA TCAGCAGGAG AAGCCCAGCT GATCTTTTTT 420AATATAGTTC TTCGAACACA ATTCGTTTAT TCATATACTC TTCATAATTT ATATTTAATT 480GGTATTTTTC AATTAAATAT AAATCAAGCT CTCTTTTAGA ATCGCAATTT TTAATAAAAG 540TTAATTCGTC TTTTTCAAAA TGTGGAATTG AAAAATTTTT AAGATATTTT TTTTGAAAGC 600AAAAGTATCC TCCGCCTATC ATATAACTAG TGTTTTCAAT ATAATATTTC ATAATAACTG 660AGTTCAATAT CTTAGCTAAA ATGTCTAAAT CGATACTTTC TACTGAATTT TTTACTCCAT 720AAATTGCATA TCCATTATTA AAAAGAGCAT AATCTGTAAA ATATACAAAG TTTGGATTCA 780AAGAATTTGT AGGAAAAATT ATTTTAGGTA CATGGCTATT CAATGCTTGA GATCGCCCAT 840ATTCATACCA AATGTTAACG GTTGGTTTCC CAGCATTGCG TTTACTGAGC TCGTCTTTAA 900TAGCAATAAA ATAATTTAAA GTATTAGGGA ATTTTTCCTT CATTGAGACA ATGCTGATTG 960GTACAGCATT GCCGTTCATA TTTTCATAAG GATATATTAT TCGATTAAAT TCGTAAAAAT 1020TATTGTTAGT ATTAACTTTT TTTTCTCCAG ATCCTTTAAT AATAGGAATA GTTATTTCTT 1080TCTCTATGAG AAAAGGTGTG TCATTATACT TTTTCACGAA ATATTCTTTA TCATTATTAA 1140CTTCTTTTTT AGTATAGTCT ATTAAATAAA GTTTATCTTT TTGAGTAGCA ATACCAGTAG 1200ATATATTCAG TGTAAAAGGT TGATTTTCTA TTTTATTTAT ATTTAATAAT TCAATTTCAT 1260CTAACAAATT AATAGATTCA GGATTAACAT CATCATACCT AATTTGATCA AACTTGTTTT 1320TTAATTCTTT TTTCATTGAT ACACTATTAC TATTCGACTG TATATTTTTA TATAATATAT 1380GACTTTTTTC ACTTTTGTCT AAAAATAATA TAGCAGAATA AGTTTGAGCA TTTGAAAAAA 1440GTTGATTATC TTTAAAATCT ATTACTTTGT ATATTGATCT AGAATCTACT AAAAGAGCCC 1500GCAAACCAAA AGCAGATTTC ATTTTTAAAA GGTGATTTGG AACAATATAA CCAATCTTTC 1560CATTTTCAGA AAGAATATTT AAACTTAATT CTATAAATGC GTAAAATAAA TTATAGCTCC 1620CAGATTTGCA AGACATATAA TGTTGTTGTA AATACTTTTT TTGATTGGAG GAGAGTTCTT 1680GTATTTTTAC ATATGGAGGA TTACCGATAA TAAAATCGAT TAAAGAAAGG CTTGGTATTA 1740TATGTTTAGC ATACTTTAAT GCCGATAGTA GAAATTCGCC ACACCCACAA GAAAAATCAC 1800CAATGGAGCT TTTTTTATTA ACTGACTTTA AAGTTTCTTC AACTATAAAG TCTGAAACTA 1860GTGAGGGAGT ATATACGATT CCATTTTCTT TCTTTGAGTT CTCACTCAAA CTAGCATATA 1920AAAATTCTTC AATATTTTTA AGAGAAAAAT GAAGCTCATT TTCTTCAATA TAATTTCTTA 1980TGTCTAAATT TGAGTAGCCT AATAGCTCGT TTATCAAACT ATTTTTAAGG GATTCTATGG 2040GTATTTTTTT TTCGGTGAAG TAATTTCTTA TTATCTCGCT TAATATTTCT TTGCTTGAAT 2100ATTTATCGAG TATTTTTTTT ATAAACTCTA TATTTGTTTG TTTATCTATA ACCTCAAGCA 2160TAATAGCACC TCATTTTTAT TTAATTATAA CTCCTAGGGT TATAAAAGTC AAGTGGAAAG 2220GAGTAACATT ATGATTATTT TTGTTCTTAA CGAACGGCTA AAAGAACTAA ATATATCACA 2280AAATAAGTTT GCGAAGCAAT CACATATTAG GCCGATACAA TAAATGATAT CTGCAATAAC 2340AGTACTAAAA GAATAGAAGT TTCAACTATC AACAAAATAC TAATTCAATT AAATAAGATA 2400GGTATTCGTA AATACTCTAT TGAAGACATA ATAAAATATA AGCATGAATA AGGAGATTTT 2460CAT ATG AAT ATA GAT CAA GTT GCA AAT AAA ATG AAA AGG GAT TTA GAA 2508 MetAsn Ile Asp Gln Val Ala Asn Lys Met Lys Arg Asp Leu Glu 305 310 315 CTAGCT ATT ACT GAT CAA ATA GTT GAC GGT TCT AAA GTA AAT AAA AAA 2556 Leu AlaIle Thr Asp Gln Ile Val Asp Gly Ser Lys Val Asn Lys Lys 320 325 330 335GGG AAA TTA TTT TTA AAT GGA GCA GAA GCA AAA CAA TCT TTA ATT AGA 2604 GlyLys Leu Phe Leu Asn Gly Ala Glu Ala Lys Gln Ser Leu Ile Arg 340 345 350TCT AGT AAA CTT ATT AAT TAT GTT CAC GAG TTT GTA AAA CAT GAA CTA 2652 SerSer Lys Leu Ile Asn Tyr Val His Glu Phe Val Lys His Glu Leu 355 360 365ATA AGA AAT AGT GTT GAA GAA TCT CTG ATA TTC CCC CCA TTA GGT CAG 2700 IleArg Asn Ser Val Glu Glu Ser Leu Ile Phe Pro Pro Leu Gly Gln 370 375 380ACA AAC CCT GAA ATA AAA CTT ACT GGT ATG TTT AAA CAA AAG GAT CAA 2748 ThrAsn Pro Glu Ile Lys Leu Thr Gly Met Phe Lys Gln Lys Asp Gln 385 390 395GAT GTT TGT GTA AAG CCT CAG GGA GTT TTA CCC GAA AGA ACT TTA ATT 2796 AspVal Cys Val Lys Pro Gln Gly Val Leu Pro Glu Arg Thr Leu Ile 400 405 410415 GGA TGG GGA CCT ATG ATA AAT TCG GGA TTA TAC TGT GAT TAT GGT CGC 2844Gly Trp Gly Pro Met Ile Asn Ser Gly Leu Tyr Cys Asp Tyr Gly Arg 420 425430 GCT TAT GCA GAA AGA GTA TTA TCT ATC AAT GTA AGA AGT CAA TTA AGT 2892Ala Tyr Ala Glu Arg Val Leu Ser Ile Asn Val Arg Ser Gln Leu Ser 435 440445 AGT CTA GAT AAA AAT TCT GAT ACG TTA TTT GAG CGG ATG TTT GCA GAA 2940Ser Leu Asp Lys Asn Ser Asp Thr Leu Phe Glu Arg Met Phe Ala Glu 450 455460 GCA TTA AAT TTA CAC GAG TTG TAT CCA AAA ATA GTT ATG GGA GAA GTA 2988Ala Leu Asn Leu His Glu Leu Tyr Pro Lys Ile Val Met Gly Glu Val 465 470475 TAT GTT ATT CCA GTT TAT GAA TAC GAC GAC CAA GCA ATG ATA AAT AAT 3036Tyr Val Ile Pro Val Tyr Glu Tyr Asp Asp Gln Ala Met Ile Asn Asn 480 485490 495 CAA GTT AAG TTC AAG TCA AGA AGA ACA AAT TTA GAA AAA TAC ATT AAT3084 Gln Val Lys Phe Lys Ser Arg Arg Thr Asn Leu Glu Lys Tyr Ile Asn 500505 510 TTT TTC TAT TAT TTA AGT GGC AGA GAT GAA CAG GAT CTT GAA GAA GAC3132 Phe Phe Tyr Tyr Leu Ser Gly Arg Asp Glu Gln Asp Leu Glu Glu Asp 515520 525 AAA CAA AAG TAC GAA AGG TGC GCA TTG GTT ATA ATA GAT TTT AGA GGA3180 Lys Gln Lys Tyr Glu Arg Cys Ala Leu Val Ile Ile Asp Phe Arg Gly 530535 540 GAT CAA GCC AAA GTC TAT AAA AAT ACT GCA GAG TTA AAA GCT AGG GGC3228 Asp Gln Ala Lys Val Tyr Lys Asn Thr Ala Glu Leu Lys Ala Arg Gly 545550 555 TTA GTC AGA AAT GAT TTT GAG GTT GAG TTA GCA GAA CTT TCA ACG GAT3276 Leu Val Arg Asn Asp Phe Glu Val Glu Leu Ala Glu Leu Ser Thr Asp 560565 570 575 AAA TTT ATT GAA GAC TTA TTA CTT ATT TAT AAT AAT AGA TTT CCTGGT 3324 Lys Phe Ile Glu Asp Leu Leu Leu Ile Tyr Asn Asn Arg Phe Pro Gly580 585 590 TCT GTT GCG AAG TTT GAA AAT CAA ACG CGC CCT CTC TGAACTCCAA3370 Ser Val Ala Lys Phe Glu Asn Gln Thr Arg Pro Leu 595 600 ATATCTTAGGCTGGTATTCC CATTAATACC TTGATTTCAG TAGACACCGA AAAGCCGAAG 3430 AGAGTTCCATTTCTTCGGTT CTTTTTATAT ATTCCTCGAA TGGTCTGCAT CCCCTTAATC 3490 GTGGAAGAGGCTGTACGGAG ACTTTGATAA AATTTATTCC GTCGTTTAAT AGGTCGATGG 3550 TCTTGTTCTATTAAATTGTT AAGATACTTC ACAGTTCGGT GCTCTGTCTT AGTATATAAA 3610 CCCACACTCTGTAACTTTCT AAAGCGGAGC CAAGAGAAGG TGCTTTATCG TGCAATTGAT 3670 GCGGACGATCAAAATATTAT TGGGAATACC TGCTTA 3706 580 amino acids amino acid linearprotein not provided 10 Met Leu Glu Val Ile Asp Lys Gln Thr Asn Ile GluPhe Ile Lys Lys 1 5 10 15 Ile Leu Asp Lys Tyr Ser Ser Lys Glu Ile LeuSer Glu Ile Ile Arg 20 25 30 Asn Tyr Phe Thr Glu Lys Lys Ile Pro Ile GluSer Leu Lys Asn Ser 35 40 45 Leu Ile Asn Glu Leu Leu Gly Tyr Ser Asn LeuAsp Ile Arg Asn Tyr 50 55 60 Ile Glu Glu Asn Glu Leu His Phe Ser Leu LysAsn Ile Glu Glu Phe 65 70 75 80 Leu Tyr Ala Ser Leu Ser Glu Asn Ser LysLys Glu Asn Gly Ile Val 85 90 95 Tyr Thr Pro Ser Leu Val Ser Asp Phe IleVal Glu Glu Thr Leu Lys 100 105 110 Ser Val Asn Lys Lys Ser Ser Ile GlyAsp Phe Ser Cys Gly Cys Gly 115 120 125 Glu Phe Leu Leu Ser Ala Leu LysTyr Ala Lys His Ile Ile Pro Ser 130 135 140 Leu Ser Leu Ile Asp Phe IleIle Gly Asn Pro Pro Tyr Val Lys Ile 145 150 155 160 Gln Glu Leu Ser SerAsn Gln Lys Lys Tyr Leu Gln Gln His Tyr Met 165 170 175 Ser Cys Lys SerGly Ser Tyr Asn Leu Phe Tyr Ala Phe Ile Glu Leu 180 185 190 Ser Leu AsnIle Leu Ser Glu Asn Gly Lys Ile Gly Tyr Ile Val Pro 195 200 205 Asn HisLeu Leu Lys Met Lys Ser Ala Phe Gly Leu Arg Ala Leu Leu 210 215 220 ValAsp Ser Arg Ser Ile Tyr Lys Val Ile Asp Phe Lys Asp Asn Gln 225 230 235240 Leu Phe Ser Asn Ala Gln Thr Tyr Ser Ala Ile Leu Phe Leu Asp Lys 245250 255 Ser Glu Lys Ser His Ile Leu Tyr Lys Asn Ile Gln Ser Asn Ser Asn260 265 270 Ser Val Ser Met Lys Lys Glu Leu Lys Asn Lys Phe Asp Gln IleArg 275 280 285 Tyr Asp Asp Val Asn Pro Glu Ser Ile Asn Leu Leu Asp GluIle Glu 290 295 300 Leu Leu Asn Ile Asn Lys Ile Glu Asn Gln Pro Phe ThrLeu Asn Ile 305 310 315 320 Ser Thr Gly Ile Ala Thr Gln Lys Asp Lys LeuTyr Leu Ile Asp Tyr 325 330 335 Thr Lys Lys Glu Val Asn Asn Asp Lys GluTyr Phe Val Lys Lys Tyr 340 345 350 Asn Asp Thr Pro Phe Leu Ile Glu LysGlu Ile Thr Ile Pro Ile Ile 355 360 365 Lys Gly Ser Gly Glu Lys Lys ValAsn Thr Asn Asn Asn Phe Tyr Glu 370 375 380 Phe Asn Arg Ile Ile Tyr ProTyr Glu Asn Met Asn Gly Asn Ala Val 385 390 395 400 Pro Ile Ser Ile ValSer Met Lys Glu Lys Phe Pro Asn Thr Leu Asn 405 410 415 Tyr Phe Ile AlaIle Lys Asp Glu Leu Ser Lys Arg Asn Ala Gly Lys 420 425 430 Pro Thr ValAsn Ile Trp Tyr Glu Tyr Gly Arg Ser Gln Ala Leu Asn 435 440 445 Ser HisVal Pro Lys Ile Ile Phe Pro Thr Asn Ser Leu Asn Pro Asn 450 455 460 PheVal Tyr Phe Thr Asp Tyr Ala Leu Phe Asn Asn Gly Tyr Ala Ile 465 470 475480 Tyr Gly Val Lys Asn Ser Val Glu Ser Ile Asp Leu Asp Ile Leu Ala 485490 495 Lys Ile Leu Asn Ser Val Ile Met Lys Tyr Tyr Ile Glu Asn Thr Ser500 505 510 Tyr Met Ile Gly Gly Gly Tyr Phe Cys Phe Gln Lys Lys Tyr LeuLys 515 520 525 Asn Phe Ser Ile Pro His Phe Glu Lys Asp Glu Leu Thr PheIle Lys 530 535 540 Asn Cys Asp Ser Lys Arg Glu Leu Asp Leu Tyr Leu IleGlu Lys Tyr 545 550 555 560 Gln Leu Asn Ile Asn Tyr Glu Glu Tyr Met AsnLys Arg Ile Val Phe 565 570 575 Glu Glu Leu Tyr 580 299 amino acidsamino acid linear protein not provided 11 Met Asn Ile Asp Gln Val AlaAsn Lys Met Lys Arg Asp Leu Glu Leu 1 5 10 15 Ala Ile Thr Asp Gln IleVal Asp Gly Ser Lys Val Asn Lys Lys Gly 20 25 30 Lys Leu Phe Leu Asn GlyAla Glu Ala Lys Gln Ser Leu Ile Arg Ser 35 40 45 Ser Lys Leu Ile Asn TyrVal His Glu Phe Val Lys His Glu Leu Ile 50 55 60 Arg Asn Ser Val Glu GluSer Leu Ile Phe Pro Pro Leu Gly Gln Thr 65 70 75 80 Asn Pro Glu Ile LysLeu Thr Gly Met Phe Lys Gln Lys Asp Gln Asp 85 90 95 Val Cys Val Lys ProGln Gly Val Leu Pro Glu Arg Thr Leu Ile Gly 100 105 110 Trp Gly Pro MetIle Asn Ser Gly Leu Tyr Cys Asp Tyr Gly Arg Ala 115 120 125 Tyr Ala GluArg Val Leu Ser Ile Asn Val Arg Ser Gln Leu Ser Ser 130 135 140 Leu AspLys Asn Ser Asp Thr Leu Phe Glu Arg Met Phe Ala Glu Ala 145 150 155 160Leu Asn Leu His Glu Leu Tyr Pro Lys Ile Val Met Gly Glu Val Tyr 165 170175 Val Ile Pro Val Tyr Glu Tyr Asp Asp Gln Ala Met Ile Asn Asn Gln 180185 190 Val Lys Phe Lys Ser Arg Arg Thr Asn Leu Glu Lys Tyr Ile Asn Phe195 200 205 Phe Tyr Tyr Leu Ser Gly Arg Asp Glu Gln Asp Leu Glu Glu AspLys 210 215 220 Gln Lys Tyr Glu Arg Cys Ala Leu Val Ile Ile Asp Phe ArgGly Asp 225 230 235 240 Gln Ala Lys Val Tyr Lys Asn Thr Ala Glu Leu LysAla Arg Gly Leu 245 250 255 Val Arg Asn Asp Phe Glu Val Glu Leu Ala GluLeu Ser Thr Asp Lys 260 265 270 Phe Ile Glu Asp Leu Leu Leu Ile Tyr AsnAsn Arg Phe Pro Gly Ser 275 280 285 Val Ala Lys Phe Glu Asn Gln Thr ArgPro Leu 290 295 2355 base pairs nucleic acid double linear DNA (genomic)NO NO Lactococcus lactis subsp. cremoris W39 CDS 744..1283 experimental/codon_start= 744 /product= “LlaDII restriction endonuclease” /evidence=EXPERIMENTAL /gene= “ORF” /number= 1 /standard_name= “Gene coding forR.LlaDII” /label= r-llaDII /note= “The first ten amino acids in thissequence may be doubtful. However, from base 773 this reading framegives a high homology with the Bsp6I endonuclease” CDS 1392..2342experimental /codon_start= 1392 /product= “LlaDII methylase” /evidence=EXPERIMENTAL /gene= “ORF” /number= 2 /standard_name= “Gene coding forM.LlaDII” /label= m-llaDII /note= “The sequence shows 60 % identity and76 % similarity with the Bsp6I methylase.” 12 CTGCAGAAAA AAAAGTAATTGGTCGTAACG AAACACGATT ATTTTCAGAC GAGCAACTAA 60 ATCACTTATC TATTGAAGTTGAACCTTATC TATTAAAGCA AGGAAATGTA GATATAGAAG 120 AATTAGAAGA ATATCGTCAAAACTTTGAAA ACTATATAGA AGAAGTAAGA AATCAGACAA 180 ATGAAAGTTA TCAGCAACAACTTGAAGCAG AACAATACGA ACCGCCAAAA GTTACAAAAA 240 GGGAAGCCAT GGAAGTCATGCTGACCGCTC TATTTGAAAA ATTTTTTGAA CCGCTCGACA 300 TTGAGCAATG GAACAAAGATAAAGCGACAA CTCATTTTTC AGAATTATCA GATATGACTG 360 ATACTGATTA TATACTTGCTTGTATGAGAT TAAACAATCC TACAACTTCA TACACAAAAG 420 AAAATGATAT AATATAGATAAAATTTAGAT ATAAAAGGAA AAACGATTAG AAAGCTTTTC 480 TTTTTTATGT CTAATTATTTGATAATAGTC CACTTTAGCG AGCTTCGCAT TGTTTTAATT 540 GTCTGATAAT TAAGAATTACAAGGCAACAA CATCTTTTAT AGATATACCA ATTACGCTTT 600 GCAAGCGGAC TGCTTCTGTCGTCAAGCAGA CCAACGAGCA TAAACAAAAA GACTTGCGAC 660 ACTACCTTAT TTCTTTTCTTTAGAAATCTC TATGGATATG ATAAAATTCT ACTTAGGGGA 720 TAAAACAACT CCAAGGTGATTTA TTG GCT TAT AAA AAG TTT GGC TAT ATT 770 Leu Ala Tyr Lys Lys Phe GlyTyr Ile 300 305 GAA ATT GAT GAT GCC AGA ATA GAT GCA ACT TGT GAT GCT TACTTT AAA 818 Glu Ile Asp Asp Ala Arg Ile Asp Ala Thr Cys Asp Ala Tyr PheLys 310 315 320 TGG AAA GAC CTA AAC TCC TAT ATT AAA AAC ACT AGT TCT CGAGGA ATG 866 Trp Lys Asp Leu Asn Ser Tyr Ile Lys Asn Thr Ser Ser Arg GlyMet 325 330 335 340 AAT ATG CCT GAT GCA ATT AGT GAA CCT ATG GGG TGT TATTGC TTA GGA 914 Asn Met Pro Asp Ala Ile Ser Glu Pro Met Gly Cys Tyr CysLeu Gly 345 350 355 TAT CTA TGG AAT AGG GGC AGT GAA GTC GGT GAT GCA ACAGAC CCA ATC 962 Tyr Leu Trp Asn Arg Gly Ser Glu Val Gly Asp Ala Thr AspPro Ile 360 365 370 ACA AAT AAA AAA ATT GAG TTT AAG GCA ACA TCA AAG TTTGAA GGG GAT 1010 Thr Asn Lys Lys Ile Glu Phe Lys Ala Thr Ser Lys Phe GluGly Asp 375 380 385 TTA TCT TCT TTC GGA CCT AAA ACA GTC TTT GAT AAT TTAGTA TTT CTG 1058 Leu Ser Ser Phe Gly Pro Lys Thr Val Phe Asp Asn Leu ValPhe Leu 390 395 400 AGA TTT TAT CTT GAT GAA AAT AAA TTA TAT ATT TAT GATTTA AAC ATT 1106 Arg Phe Tyr Leu Asp Glu Asn Lys Leu Tyr Ile Tyr Asp LeuAsn Ile 405 410 415 420 AAT TCA GAA GAG TTT GAG AAA TAT CCA GCA AAT AAGACT CAA ACT ATA 1154 Asn Ser Glu Glu Phe Glu Lys Tyr Pro Ala Asn Lys ThrGln Thr Ile 425 430 435 CAA GAA CAA AAA GCT GTT GGA AGG CGT CCT CAC GTGAGT TTA CAA TCT 1202 Gln Glu Gln Lys Ala Val Gly Arg Arg Pro His Val SerLeu Gln Ser 440 445 450 TTG TTT GTA GAC GCA AAA AAC TTA CAA CCA GAT ATTATT TTT GAT ATT 1250 Leu Phe Val Asp Ala Lys Asn Leu Gln Pro Asp Ile IlePhe Asp Ile 455 460 465 AGA CGA TGT CGA ATT ATC GAA GAT AAT AGA CACTAAACTGAAA GGGGAGTTGT 1303 Arg Arg Cys Arg Ile Ile Glu Asp Asn Arg His470 475 TTTATCTCCC TTTTCATCAT ATTAAAATTG CGGCGTGCCG CTTTTTGTTGTATACTATAT 1363 ATCATACTTG ATTTATAGGA GAATATTT ATG TTG AAA ATT GCT TCTTTT TTC 1415 Met Leu Lys Ile Ala Ser Phe Phe 1 5 GCC GGA GTT GGC GGA ATTGAT TTA GGT TTT GAA AAT GCA GGT TTC AAA 1463 Ala Gly Val Gly Gly Ile AspLeu Gly Phe Glu Asn Ala Gly Phe Lys 10 15 20 ACA ATA TAT GCT AAT GAA TTTGAT AAT TAT GCT GCT GAT ACT TTT GAA 1511 Thr Ile Tyr Ala Asn Glu Phe AspAsn Tyr Ala Ala Asp Thr Phe Glu 25 30 35 40 ATG AAC TTT GAC GTT AAG GTAGAC CGA CGT GAT ATA AAT GAT GTA CAA 1559 Met Asn Phe Asp Val Lys Val AspArg Arg Asp Ile Asn Asp Val Gln 45 50 55 GCT GAT GAA ATA CCA GAT TTT GATATT ATG TTA GCA GGT TTT CCT TGC 1607 Ala Asp Glu Ile Pro Asp Phe Asp IleMet Leu Ala Gly Phe Pro Cys 60 65 70 CAA GCC TTT TCT ATT GCT GGT TAT CGTCAA GGC TTT AAC GAT GAA CAA 1655 Gln Ala Phe Ser Ile Ala Gly Tyr Arg GlnGly Phe Asn Asp Glu Gln 75 80 85 GGT CGA GGT AAT CTT TTT TTT GAA CTT GTTCGT ATT TTA GAA ACA AAA 1703 Gly Arg Gly Asn Leu Phe Phe Glu Leu Val ArgIle Leu Glu Thr Lys 90 95 100 AAA CCT CGT GTT GCA TTC TTT GAA AAT GTTAAA AAT CTT GTT TCT CAC 1751 Lys Pro Arg Val Ala Phe Phe Glu Asn Val LysAsn Leu Val Ser His 105 110 115 120 GAT AGC GGG AAC ACA TTT AGA GTT ATTTGT TCT GAG TTA GAA AGA CTA 1799 Asp Ser Gly Asn Thr Phe Arg Val Ile CysSer Glu Leu Glu Arg Leu 125 130 135 GGG TAC AAG TAT CTT TTT CAA GTG TTTAAT GCT TCT GAA TAT GGA AAT 1847 Gly Tyr Lys Tyr Leu Phe Gln Val Phe AsnAla Ser Glu Tyr Gly Asn 140 145 150 ATA CCT CAA AAT AGA GAA CGT ATC TATATT GTT GCT TTC AAA AAT AAA 1895 Ile Pro Gln Asn Arg Glu Arg Ile Tyr IleVal Ala Phe Lys Asn Lys 155 160 165 AAA GAT TAT GCA AAT TTT GAA CTA CCAAAA TCT ATA CCT TTA AAA ACA 1943 Lys Asp Tyr Ala Asn Phe Glu Leu Pro LysSer Ile Pro Leu Lys Thr 170 175 180 ACG ATT CAC GAT GTT ATT GAT TTT TCTAAA AAA CAA GAC GAT AAG TTC 1991 Thr Ile His Asp Val Ile Asp Phe Ser LysLys Gln Asp Asp Lys Phe 185 190 195 200 TAC TAT ACC TCT GAA AAG AAT AAATTT TTT GAT GAG TTA AAA GAA AAT 2039 Tyr Tyr Thr Ser Glu Lys Asn Lys PhePhe Asp Glu Leu Lys Glu Asn 205 210 215 ATG ACT AAT CAC GAC ACT ACA TATCAG TGG CGT AGA GTT TAT GTA AGA 2087 Met Thr Asn His Asp Thr Thr Tyr GlnTrp Arg Arg Val Tyr Val Arg 220 225 230 GAA AAC AAA AGT AAT TTA GTA CCAACA CTA ACG GCT AAT ATG GGA ACA 2135 Glu Asn Lys Ser Asn Leu Val Pro ThrLeu Thr Ala Asn Met Gly Thr 235 240 245 GGT GGG CAT AAT GTG CCT ATA ATCCTT ACA TAT AGC GGA GAT ATT CGT 2183 Gly Gly His Asn Val Pro Ile Ile LeuThr Tyr Ser Gly Asp Ile Arg 250 255 260 AAA TTA ACA CCA AGA GAA TGC TTTAAC GTT CAA GGT TTC CCA AAA GAA 2231 Lys Leu Thr Pro Arg Glu Cys Phe AsnVal Gln Gly Phe Pro Lys Glu 265 270 275 280 TAT AAA CTT CCA AAC CAA AGTAAT GGG AGA TTA TAT AAA CAA GCA GGA 2279 Tyr Lys Leu Pro Asn Gln Ser AsnGly Arg Leu Tyr Lys Gln Ala Gly 285 290 295 AAC AGT GTT GTA GTA CCA GTTATA GAA AGA ATT GCA AAA AAT CTT GCA 2327 Asn Ser Val Val Val Pro Val IleGlu Arg Ile Ala Lys Asn Leu Ala 300 305 310 GAT ACT ATA GTC GAATAACTATGAA TTC 2355 Asp Thr Ile Val Glu 315 180 amino acids amino acidlinear protein not provided 13 Leu Ala Tyr Lys Lys Phe Gly Tyr Ile GluIle Asp Asp Ala Arg Ile 1 5 10 15 Asp Ala Thr Cys Asp Ala Tyr Phe LysTrp Lys Asp Leu Asn Ser Tyr 20 25 30 Ile Lys Asn Thr Ser Ser Arg Gly MetAsn Met Pro Asp Ala Ile Ser 35 40 45 Glu Pro Met Gly Cys Tyr Cys Leu GlyTyr Leu Trp Asn Arg Gly Ser 50 55 60 Glu Val Gly Asp Ala Thr Asp Pro IleThr Asn Lys Lys Ile Glu Phe 65 70 75 80 Lys Ala Thr Ser Lys Phe Glu GlyAsp Leu Ser Ser Phe Gly Pro Lys 85 90 95 Thr Val Phe Asp Asn Leu Val PheLeu Arg Phe Tyr Leu Asp Glu Asn 100 105 110 Lys Leu Tyr Ile Tyr Asp LeuAsn Ile Asn Ser Glu Glu Phe Glu Lys 115 120 125 Tyr Pro Ala Asn Lys ThrGln Thr Ile Gln Glu Gln Lys Ala Val Gly 130 135 140 Arg Arg Pro His ValSer Leu Gln Ser Leu Phe Val Asp Ala Lys Asn 145 150 155 160 Leu Gln ProAsp Ile Ile Phe Asp Ile Arg Arg Cys Arg Ile Ile Glu 165 170 175 Asp AsnArg His 180 317 amino acids amino acid linear protein not provided 14Met Leu Lys Ile Ala Ser Phe Phe Ala Gly Val Gly Gly Ile Asp Leu 1 5 1015 Gly Phe Glu Asn Ala Gly Phe Lys Thr Ile Tyr Ala Asn Glu Phe Asp 20 2530 Asn Tyr Ala Ala Asp Thr Phe Glu Met Asn Phe Asp Val Lys Val Asp 35 4045 Arg Arg Asp Ile Asn Asp Val Gln Ala Asp Glu Ile Pro Asp Phe Asp 50 5560 Ile Met Leu Ala Gly Phe Pro Cys Gln Ala Phe Ser Ile Ala Gly Tyr 65 7075 80 Arg Gln Gly Phe Asn Asp Glu Gln Gly Arg Gly Asn Leu Phe Phe Glu 8590 95 Leu Val Arg Ile Leu Glu Thr Lys Lys Pro Arg Val Ala Phe Phe Glu100 105 110 Asn Val Lys Asn Leu Val Ser His Asp Ser Gly Asn Thr Phe ArgVal 115 120 125 Ile Cys Ser Glu Leu Glu Arg Leu Gly Tyr Lys Tyr Leu PheGln Val 130 135 140 Phe Asn Ala Ser Glu Tyr Gly Asn Ile Pro Gln Asn ArgGlu Arg Ile 145 150 155 160 Tyr Ile Val Ala Phe Lys Asn Lys Lys Asp TyrAla Asn Phe Glu Leu 165 170 175 Pro Lys Ser Ile Pro Leu Lys Thr Thr IleHis Asp Val Ile Asp Phe 180 185 190 Ser Lys Lys Gln Asp Asp Lys Phe TyrTyr Thr Ser Glu Lys Asn Lys 195 200 205 Phe Phe Asp Glu Leu Lys Glu AsnMet Thr Asn His Asp Thr Thr Tyr 210 215 220 Gln Trp Arg Arg Val Tyr ValArg Glu Asn Lys Ser Asn Leu Val Pro 225 230 235 240 Thr Leu Thr Ala AsnMet Gly Thr Gly Gly His Asn Val Pro Ile Ile 245 250 255 Leu Thr Tyr SerGly Asp Ile Arg Lys Leu Thr Pro Arg Glu Cys Phe 260 265 270 Asn Val GlnGly Phe Pro Lys Glu Tyr Lys Leu Pro Asn Gln Ser Asn 275 280 285 Gly ArgLeu Tyr Lys Gln Ala Gly Asn Ser Val Val Val Pro Val Ile 290 295 300 GluArg Ile Ala Lys Asn Leu Ala Asp Thr Ile Val Glu 305 310 315

What is claimed is:
 1. A plasmid-derived type IIrestriction-modification (R-M) system, termed LlaDII, from Lactococcuslactis subsp. cremoris W39, said system encoding at least one methylaseand a restriction endonuclease, characterized in that the systemcomprises i) an open reading frame, termed ORF1, from about nucleotide743 to nucleotide 1282 in the enclosed SEQ ID No. 12, coding for arestriction endonuclease, termed R.LlaDII, having the amino acidsequence essentially as shown in the enclosed SEQ ID No. 13 and with therecognition sequence 5′-GC↓NGC-3′, and ii) an open reading frame, termedORF2, from nucleotide 1391 to nucleotide 2341 in the enclosed SEQ ID No.12, coding for a methylase, termed M.LlaDII, having the amino acidsequence shown in the enclosed SEQ ID No.
 14. 2. A DNA fragment codingfor a restriction endonuclease, termed R.LlaDII, said fragmentcomprising the DNA sequence from nucleotide 743 to nucleotide 1282 inthe enclosed SEQ ID No.12.
 3. A DNA fragment coding for a methylase,termed M.LlaDII, said fragment comprising the DNA sequence fromnucleotide 1391 to nucleotide 2341 in the enclosed SEQ ID No.
 12. 4. ADNA cassette comprising the R-M system and DNA fragments according toany one of the preceding claims in combination with DNA encoding otherphage resistance mechanisms selected from the group consisting ofadsorption blocking, abortive infection and R-M systems.
 5. A cloningvector including DNA of the R-M system according to claim 1 and a DNAcassette according to claim
 4. 6. A cloning vector according to claim 5which is the plasmid pCAD1 introduced in Lactococcus lactis subsp.cremoris LM2301 and deposited under the accession number LMG P-16901. 7.An expression vector including DNA of the R-M system according to claim1 and a DNA cassette according to claim 4 under the control of apromoter capable of providing expression thereof in a host cell.
 8. Anexpression vector according to claim 7 wherein said DNA is under thecontrol of a promoter capable of providing expression thereof in aGram-positive bacterium.
 9. An expression vector according to claim 8wherein said DNA is under the control of a promoter capable of providingexpression thereof in a lactic acid bacterium.
 10. An expression vectoraccording to claim 9 wherein said DNA is under the control of a promotercapable of providing expression thereof in Lactococcus lactis.
 11. Acloning vector including DNA selected from the group consisting of DNAfragments according to claims 2 and 3 and a DNA cassette according toclaim
 4. 12. A cloning vector according to claim 11 comprising theplasmid pCAD I introduced in Lactococcus lactis subsp. cremoris LM2301and deposited under the accession number LMG P-16901.
 13. An expressionvector including DNA selected from the group consisting of DNA fragmentsaccording to claims 2 and 3 and a DNA cassette according to claim 4,under the control of a promoter capable of providing expression of saidDNA in a host cell.
 14. An expression vector according to claim 13wherein said DNA is under the control of a promoter capable of providingexpression of said DNA in a Gram-positive bacterium.
 15. An expressionvector according to claim 14 wherein said DNA is under the control of apromoter capable of providing expression of said DNA in a lactic acidbacterium.
 16. An expression vector according to claim 15 wherein saidDNA is under the control of a promoter capable of providing expressionof said DNA in Lactococcus lactis.
 17. A method of conferring increasedvirus resistance on a cell wherein said cell is transformed with anexpression vector according to claim
 7. 18. A method of conferringincreased phage resistance on a Gram-positive bacterium wherein saidbacterium is transformed with an expression vector according to claim 8.19. A method of conferring increased phage resistance on a lactic acidbacterium wherein said bacterium is transformed with an expressionvector according to claim
 9. 20. A method of conferring increased phageresistance on a Lactococcus lactis strain wherein said strain istransformed with an expression vector according to claim
 10. 21. A cellwhich carries an expression vector according to claim
 7. 22. AGram-positive bacterium which carries an expression vector according toclaim
 8. 23. A lactic acid bacterium which carries an expression vectoraccording to claim
 9. 24. A Lactococcus lactis strain which carries anexpression vector according to claim
 10. 25. A method of conferringincreased virus resistance on a cell which comprises transforming saidcell with an expression vector according to claim
 13. 26. A method ofconferring increased phage resistance on a Gram-positive bacterium whichcomprises transforming said bacterium with an expression vectoraccording to claim
 14. 27. A method of conferring increased phageresistance on a lactic acid bacterium which comprises transforming saidbacterium with an expression vector according to claim
 15. 28. A methodof conferring increased phage resistance on a Lactococcus lactis strainwhich comprises transforming said strain with an expression vectoraccording to claim
 16. 29. A cell which carries an expression vectoraccording to claim
 13. 30. A Gram-positive bacterium which carries anexpression vector according to claim
 14. 31. A lactic acid bacteriumwhich carries an expression vector according to claim
 15. 32. ALactococcus lactis strain which carries an expression vector accordingto claim
 16. 33. A plasmid comprising the DNA sequence encoding the R-Msystem of claim
 1. 34. A plasmid comprising the DNA sequence encodingthe DNA cassette of claim
 4. 35. A restriction endonuclease, termedR.LlaDII, with the recognition sequence 5′-GC↓NGC-3′, said endonucleasehaving the amino acid sequence essentially as shown in the enclosed SEQID No.
 13. 36. A methylase, termed M.LlaDII, having the amino acidsequence shown in the enclosed SEQ ID No. 14.